Methods of treating cancer patients with farnesyltransferase inhibitors

ABSTRACT

The present invention relates to the field of molecular biology and cancer biology. Specifically, the present invention relates to methods of treating a subject with a farnesyltransferase inhibitor (FTI) that include determining whether the subject is likely to be responsive to the FTI treatment based on HRAS mutation status in the subject.

FIELD

The present invention relates to the field of molecular biology andcancer biology. Provided herein are methods of using HRAS mutationsbiomarkers for predicting clinical sensitivity and therapeutic responseto treatment with a farnesyltransferase inhibitor in a subject having asquamous cell head and neck cancer or squamous cell lung cancer that hasnot yet been treated with an EGFR inhibitor or that is refractory totreatment with an EGFR inhibitor. Further provided herein are kits forcarrying out these methods.

BACKGROUND

Stratification of patient populations to improve therapeutic responserate is increasingly valuable in the clinical management of cancerpatients. Farnesyltransferase inhibitors (FTI) are therapeutic agentsthat have utility in the treatment of cancers, such as leukemia,lymphoma and certain solid tumors. However, different patients mayrespond differently to an FTI treatment. Therefore, methods to predictthe responsiveness of a cancer patient to an FTI treatment, or methodsto select patients for an FTI treatment represent unmet needs. Themethods and compositions of the present invention meet these needs andprovide other related advantages.

SUMMARY OF THE INVENTION

Provided herein are methods for population selection of head and neckcancer patients for treatment with an FTI. The methods provided hereinare based, in part, on the discovery that the mutant status of HRASand/or resistance of said cancer to EGFR inhibitors can be used topredict responsiveness of a head and neck cancer patient to an FTItreatment.

Provided herein are methods of treating EGFR inhibitor-refractorysquamous cell carcinoma of the head and neck (SCCHN), wherein the SCCHNhas an HRAS mutation, comprising administering to the subject afarnesyltransferase inhibitor (FTI). In certain embodiments, said HRASmutation comprises an amino acid substitution at a codon selected from agroup consisting of G12, G13, Q61, Q22, K117, A146 and any combinationthereof. In certain embodiments, said SCCHN does not have K-Ras mutationor N-Ras mutation. In certain embodiments, said SCCHN has an amplifiedHRAS gene or overexpresses the HRAS mRNA and/or protein. In certainembodiments, said SCCHN has wild type K-Ras and wild type N-Ras. Incertain embodiments, said SCCHN is HPV negative. In certain embodiments,said SCCHN is HPV positive. In certain embodiments, said SCCHN is at anadvanced stage or metastatic. In certain embodiments, said SCCHN isrelapsed SCCHN. In specific embodiments, the SCCHN is SCCHN of thetrachea. In specific embodiments, the SCCHN is SCCHN of the maxilla. Inspecific embodiments, the SCCHN is SCCHN of the oral cavity. In certainembodiments, the EGFR inhibitor is cetuximab. In certain embodiments,the FTI is tipifarnib.

Provided herein are methods of treating a squamous cell carcinoma of thehead and neck (SCCHN or HNSCC) in a subject, wherein the SCCHN isrefractory to an EGFR inhibitor, comprising (a) determining the presenceor absence of a HRAS mutation in a sample from said subject, andsubsequently (b) administering a therapeutically effective amount of afarnesyltransferase inhibitor (FTI) to said subject if said sample isdetermined to have a HRAS mutation. Also provided herein are methods oftreating a squamous cell carcinoma of the head and neck (SCCHN) in asubject, wherein the subject has never been treated with an EGFRinhibitor, comprising (a) determining the presence or absence of a HRASmutation in a sample from said subject, and subsequently (b)administering a therapeutically effective amount of afarnesyltransferase inhibitor (FTI) to said subject if said sample isdetermined to have a HRAS mutation and not administering an EGFRinhibitor. In certain embodiments, said HRAS mutation comprises an aminoacid substitution at a codon selected from a group consisting of G12,G13, Q61, Q22, K117, A146, and any combination thereof. In certainembodiments, said FTI is administered in combination with chemotherapy.In certain embodiments, said chemotherapy comprises a platinum-basedtherapy, a taxane, or a combination thereof.

In certain embodiments, the methods further comprise determining thepresence or absence of a K-Ras mutation or a N-Ras mutation, whereinsaid sample does not have K-Ras mutation or N-Ras mutation. In certainembodiments, said sample has wild type K-Ras and wild type N-Ras. Inspecific embodiments, the sample is a tissue biopsy. In specificembodiments, the sample is a tumor biopsy. In specific embodiments,wherein determining the presence or absence of a Ras mutation comprisinganalyzing nucleic acids obtained from said sample.

In certain embodiments, Ras mutation is determined by sequencing,Polymerase Chain Reaction (PCR), DNA microarray, Mass Spectrometry (MS),Single Nucleotide Polymorphism (SNP) assay, denaturing high-performanceliquid chromatography (DHPLC), or Restriction Fragment LengthPolymorphism (RFLP) assay. In specific embodiments, Ras mutation isdetermined by PCR. In specific embodiments, Ras mutation is determinedby sequencing. In specific embodiments, determining the presence orabsence of a Ras mutation comprising analyzing proteins obtained fromsaid sample.

In certain embodiments, said SCCHN is HPV negative. In certainembodiments, said SCCHN is HPV positive. In certain embodiments, saidSCCHN is at an advanced stage or metastatic. In certain embodiments,said SCCHN is relapsed SCCHN. In certain embodiments, the SCCHN is SCCHNof the trachea. In certain embodiments, the SCCHN is SCCHN of themaxilla. In certain embodiments, the SCCHN is SCCHN of the oral cavity.

In certain embodiments, the EGFR inhibitor is cetuximab. In certainembodiments, the EGFR inhibitor is erlotinib. In certain embodiments,the EGFR inhibitor is gefitinib. In certain embodiments, the EGFRinhibitor is panitumumab. In certain embodiments, thefarnesyltransferase inhibitor (FTI) is tipifarnib.

In some embodiments, provided herein is a method of treating a SCCHN ina subject based on the presence of an HRAS mutation. In someembodiments, the SCCHN can be HPV negative SCCHN. In some embodiments,the SCCHN can be HPV positive SCCHN. In some embodiments, the SCCHN canbe relapsed/recurrent SCCHN. In some embodiments, the SCCHN can bemetastatic SCCHN. The methods provided herein include (a) determiningthe presence or absence of a HRAS mutation in a sample from the subjecthaving SCCHN, and subsequently (b) administering a therapeuticallyeffective amount of tipifarnib to the subject if the sample isdetermined to have a HRAS mutation. In certain embodiments, saidtipifarnib is administered in combination with chemotherapy. In certainembodiments, said chemotherapy comprises a platinum-based therapy, ataxane, or a combination thereof.

Provided herein are methods of treating EGFR inhibitor-refractory lungsquamous cell carcinoma (lung SCC), wherein the lung SCC has an HRASmutation, comprising administering to the subject a farnesyltransferaseinhibitor (FTI). In certain embodiments, said HRAS mutation comprises anamino acid substitution at a codon selected from a group consisting ofG12, G13, Q61, Q22, K117, A146 and any combination thereof. In certainembodiments, said lung SCC does not have K-Ras mutation or N-Rasmutation. In certain embodiments, said lung SCC has an amplified HRASgene or overexpresses the HRAS mRNA and/or protein. In certainembodiments, said lung SCC has wild type K-Ras and wild type N-Ras. Incertain embodiments, said lung SCC is HPV negative. In certainembodiments, said lung SCC is HPV positive. In certain embodiments, saidlung SCC is at an advanced stage or metastatic. In certain embodiments,said lung SCC is relapsed lung SCC. In certain embodiments, the EGFRinhibitor is cetuximab. In certain embodiments, the FTI is tipifarnib.

Provided herein are methods of treating a lung squamous cell carcinoma(lung SCC) in a subject, wherein the lung SCC is refractory to an EGFRinhibitor, comprising (a) determining the presence or absence of a HRASmutation in a sample from said subject, and subsequently (b)administering a therapeutically effective amount of afarnesyltransferase inhibitor (FTI) to said subject if said sample isdetermined to have a HRAS mutation. Also provided herein are methods oftreating a lung SCC in a subject, wherein the subject has never beentreated with an EGFR inhibitor, comprising (a) determining the presenceor absence of a HRAS mutation in a sample from said subject, andsubsequently (b) administering a therapeutically effective amount of afarnesyltransferase inhibitor (FTI) to said subject if said sample isdetermined to have a HRAS mutation and not administering an EGFRinhibitor. In certain embodiments, said HRAS mutation comprises an aminoacid substitution at a codon selected from a group consisting of G12,G13, Q61, Q22, K117, A146, and any combination thereof. In certainembodiments, said FTI is administered in combination with chemotherapy.In certain embodiments, said chemotherapy comprises a platinum-basedtherapy, a taxane, or a combination thereof.

In certain embodiments, the methods further comprise determining thepresence or absence of a K-Ras mutation or a N-Ras mutation, whereinsaid sample does not have K-Ras mutation or N-Ras mutation. In certainembodiments, said sample has wild type K-Ras and wild type N-Ras. Inspecific embodiments, the sample is a tissue biopsy. In specificembodiments, the sample is a tumor biopsy. In specific embodiments,wherein determining the presence or absence of a Ras mutation comprisinganalyzing nucleic acids obtained from said sample.

In certain embodiments, Ras mutation is determined by sequencing,Polymerase Chain Reaction (PCR), DNA microarray, Mass Spectrometry (MS),Single Nucleotide Polymorphism (SNP) assay, denaturing high-performanceliquid chromatography (DHPLC), or Restriction Fragment LengthPolymorphism (RFLP) assay. In specific embodiments, Ras mutation isdetermined by PCR. In specific embodiments, Ras mutation is determinedby sequencing. In specific embodiments, determining the presence orabsence of a Ras mutation comprising analyzing proteins obtained fromsaid sample.

In certain embodiments, said lung SCC is HPV negative. In certainembodiments, said lung SCC is HPV positive. In certain embodiments, saidlung SCC is at an advanced stage or metastatic. In certain embodiments,said lung SCC is relapsed lung SCC.

In certain embodiments, the EGFR inhibitor is cetuximab. In certainembodiments, the EGFR inhibitor is erlotinib. In certain embodiments,the EGFR inhibitor is gefitinib. In certain embodiments, the EGFRinhibitor is panitumumab. In certain embodiments, thefarnesyltransferase inhibitor (FTI) is tipifarnib.

In some embodiments, provided herein is a method of treating a lung SCCin a subject based on the presence of an HRAS mutation. In someembodiments, the lung SCC can be HPV negative lung SCC. In someembodiments, the lung SCC can be HPV positive lung SCC. In someembodiments, the lung SCC can be relapsed/recurrent lung SCC. In someembodiments, the lung SCC can be metastatic lung SCC. The methodsprovided herein include (a) determining the presence or absence of aHRAS mutation in a sample from the subject having lung SCC, andsubsequently (b) administering a therapeutically effective amount oftipifarnib to the subject if the sample is determined to have a HRASmutation. In certain embodiments, said tipifarnib is administered incombination with chemotherapy. In certain embodiments, said chemotherapycomprises a platinum-based therapy, a taxane, or a combination thereof.

In some embodiments, the FTI is selected from the group consisting oftipifarnib, lonafarnib (SCH-66336), CP-609,754, BMS-214662, L778123,L744823, L739749, R208176, AZD3409 and FTI-277. In some embodiments, theFTI is administered at a dose of 1-1000 mg/kg body weight. In oneembodiment, the FTI is tipifarnib. In some embodiments, tipifarnib isadministered at a dose of 200-1200 mg twice a day (“b.i.d.”). In someembodiments, tipifarnib is administered at a dose of 600 mg dailyorally. In some embodiments, tipifarnib is administered at a dose of 300mg b.i.d. orally for 3 of out of 4 weeks in repeated 4 week cycles. Insome embodiments, tipifarnib is administered at a dose of 600 mg b.i.d.orally for 3 of out of 4 weeks in repeated 4 week cycles. In someembodiments, tipifarnib is administered at a dose of 900 mg b.i.d.orally in alternate weeks (one week on, one week off) in repeated 4 weekcycles (days 1-7 and 15-21 of repeated 28-day cycles). In someembodiments, tipifarnib is administered at a dose of 1200 mg b.i.d.orally in alternate weeks (days 1-7 and 15-21 of repeated 28-daycycles). In some embodiments, tipifarnib is administered at a dose of600 mg b.i.d. orally in alternate weeks (days 1-7 and 15-21 of repeated28-day cycles). In some embodiments, tipifarnib is administered at adose of 400 mg b.i.d. orally in alternate weeks (days 1-7 and 15-21 ofrepeated 28-day cycles). In some embodiments, tipifarnib is administeredat a dose of 300 mg b.i.d. orally in alternate weeks (days 1-7 and 15-21of repeated 28-day cycles). In some embodiments, tipifarnib isadministered at a dose of 200 mg b.i.d. orally in alternate weeks (days1-7 and 15-21 of repeated 28-day cycles). In some embodiments,tipifarnib is administered at a dose of 1200 mg b.i.d. orally for days1-5 and 15-19 out of repeated 28-day cycles. In some embodiments,patients receive at least three cycles of treatment. In someembodiments, patients receive at least six cycles of treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-B. CT scan of tumor of subject 1 at (A) baseline and (B) cycle4, day 22. Tumor indicated in circle.

FIG. 2. Tumor volumes of mice in different groups during tipifarnibtreatment in HUPRIME® head and neck cancer xenograft model HN1420,wherein group 01 is the vehicle group and group 02 is the tipifarnibgroup.

FIG. 3. Tumor Volumes of mice in different groups during tipifarnibtreatment in HUPRIME® NSCLC xenograft model LU1513, wherein group 01 isthe vehicle group and group 02 is the tipifarnib group.

DETAILED DESCRIPTION 1. Definitions

As used herein, the articles “a,” “an,” and “the” refer to one or tomore than one of the grammatical object of the article. By way ofexample, a biomarker refers to one biomarker or more than onebiomarkers.

As used herein, the term “subject” refers to a mammal. A subject can bea human or a non-human mammal such as a dog, cat, bovid, equine, mouse,rat, rabbit, or transgenic species thereof. The subject can be apatient, or a cancer patient.

As used herein, the term “cancer” or “cancerous” refers to thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include, but are not limitedto, hematological cancers (e.g., multiple myeloma, lymphoma andleukemia), and solid tumors. As used herein, the term “premalignantcondition” refers to a condition associated with an increased risk ofcancer, which, if left untreated, can lead to cancer. A premalignantcondition can also refer to non-invasive cancer that have not progressedinto aggressive, invasive stage.

As used herein, the term “treat,” “treating,” and “treatment,” when usedin reference to a cancer patient, refer to an action that reduces theseverity of the cancer, or retards or slows the progression of thecancer, including (a) inhibiting the cancer growth, or arrestingdevelopment of the cancer, and (b) causing regression of the cancer, ordelaying or minimizing one or more symptoms associated with the presenceof the cancer.

As used herein, the term “determining” refers to using any form ofmeasurement to assess the presence of a substance, either quantitativelyor qualitatively. Measurement can be relative or absolute. Measuring thepresence of a substance can include determining whether the substance ispresent or absent, or the amount of the substance.

As used herein, the term “administer,” “administering,” or“administration” refers to the act of delivering, or causing to bedelivered, a compound or a pharmaceutical composition to the body of asubject by a method described herein or otherwise known in the art.Administering a compound or a pharmaceutical composition includesprescribing a compound or a pharmaceutical composition to be deliveredinto the body of a patient. Exemplary forms of administration includeoral dosage forms, such as tablets, capsules, syrups, suspensions;injectable dosage forms, such as intravenous (IV), intramuscular (IM),or intraperitoneal (IP); transdermal dosage forms, including creams,jellies, powders, or patches; buccal dosage forms; inhalation powders,sprays, suspensions, and rectal suppositories.

As used herein, the term “therapeutically effective amount” of acompound when used in connection with a disease or disorder refers to anamount sufficient to provide a therapeutic benefit in the treatment ormanagement of the disease or disorder or to delay or minimize one ormore symptoms associated with the disease or disorder. A therapeuticallyeffective amount of a compound means an amount of the compound, alone orin combination with other therapies, that provides a therapeutic benefitin the treatment or management of the disease or disorder. The termencompasses an amount that improves overall therapy, reduces or avoidssymptoms, or enhances the therapeutic efficacy of another therapeuticagent. The term also refers to the amount of a compound thatsufficiently elicits the biological or medical response of a biologicalmolecule (e.g., a protein, enzyme, RNA, or DNA), cell, tissue, system,animal, or human, which is being sought by a researcher, veterinarian,medical doctor, or clinician.

As used herein, the term “sample” refers to a material or mixture ofmaterials containing one or more components of interest. A sample from asubject refers to a sample obtained from the subject, including samplesof biological tissue or fluid origin, obtained, reached, or collected invivo or in situ. A sample can be obtained from a region of a subjectcontaining precancerous or cancer cells or tissues. Such samples can be,but are not limited to, organs, tissues, fractions and cells isolatedfrom a mammal. Exemplary samples include bone marrow, whole blood,partially purified blood, peripheral blood mononuclear cells (“PBMC”),and tissue biopsies. Exemplary samples also include cell lysate, a cellculture, a cell line, a tissue, oral tissue, gastrointestinal tissue, anorgan, an organelle, a biological fluid, a blood sample, a urine sample,a skin sample, and the like.

As used herein, the term “biomarker” refers to a gene that can be eitherpresent or absent in individual subjects, or can be present butdifferentially expressed in individual subjects. The presence abiomarker, including the expression level of the biomarker, in a samplefrom a subject can indicate the responsiveness of the subject to aparticular treatment, such as an FTI treatment.

As used herein, the term “express” or “expression” when used inconnection with a gene refers to the process by which the informationcarried by the gene becomes manifest as the phenotype, includingtranscription of the gene to a messenger RNA (mRNA), the subsequenttranslation of the mRNA molecule to a polypeptide chain and its assemblyinto the ultimate protein.

As used herein, the term “RNA product of the biomarker” refers to a RNAtranscript transcribed from a biomarker, and the term “protein productof the biomarker” refers to a protein or polypeptide translated from aRNA product of a biomarker.

As used herein, the term “expression level” of a biomarker refers to theamount or accumulation of the expression product of a biomarker, suchas, for example, the amount of a RNA product of the biomarker (the RNAlevel of the biomarker) or the amount of a protein product of thebiomarker (the protein level of the biomarker). If the biomarker is agene with more than one alleles, the expression level of a biomarkerrefers to the total amount of accumulation of the expression product ofall existing alleles for this gene, unless otherwise specified.

As used herein, the term “reference expression level” refers to apredetermined expression level of a biomarker that one can use todetermine the significance of the expression level of the biomarker in asample from a subject. A reference expression level of a biomarker canbe the expression level of the biomarker in a sample from a healthyindividual. A reference expression level of a biomarker can also be acut-off value determined by a person of ordinary skill in the artthrough statistic analysis of the expression levels of the biomarker ina sample population and the responsiveness to a treatment of theindividuals in the sample population.

As used herein, the term “responsiveness” or “responsive” when used inconnection with a treatment refers to the effectiveness of the treatmentin lessening or decreasing the symptoms of the disease being treated.For example, a cancer patient is responsive to an FTI treatment if theFTI treatment effectively inhibits the cancer growth, or arrestsdevelopment of the cancer, causes regression of the cancer, or delays orminimizes one or more symptoms associated with the presence of thecancer in this patient.

The responsiveness to a particular treatment of a cancer patient can becharacterized as a complete or partial response. “Complete response,” or“CR” refers to an absence of clinically detectable disease withnormalization of previously abnormal radiographic studies, bone marrow,and cerebrospinal fluid (CSF) or abnormal monoclonal proteinmeasurements. “Partial response,” or “PR,” refers to at least about a10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% decrease in allmeasurable tumor burden (i.e., the number of malignant cells present inthe subject, or the measured bulk of tumor masses or the quantity ofabnormal monoclonal protein) in the absence of new lesions.

A person of ordinary skill in the art would understand that clinicalstandards used to define CR, PR, or other level of patientresponsiveness to treatments can vary for different types of cancer. Forexample, for hematopoietic cancers, patient being “responsive” to aparticular treatment can be defined as patients who have a completeresponse (CR), a partial response (PR), or hematological improvement(HI) (Lancet et al., Blood 2:2 (2006)). For solid tumors, a patientbeing “responsive” to a particular treatment can be defined by RECISTcriteria (see Therasse et al., “New guidelines to evaluate the responseto treatment in solid tumors,” JNCI 92(3):205-216 (2000)). HI can bedefined as any bone marrow blast count less than 5% or a reduction inbone marrow blasts by at least half. On the other hand, patient being“not responsive” to a particular treatment can be defined as patientswho have either progressive disease (PD) or stable disease (SD).Progressive disease (PD) can be defined as either >50% increase in bonemarrow or circulating blast % from baseline, or new appearance ofcirculating blasts (on at least 2 consecutive occasions). Stable disease(SD) can be defined as any response not meeting CR, PR, HI, or PDcriteria.

As used herein, the term “likelihood” refers to the probability of anevent. A subject is “likely” to be responsive to a particular treatmentwhen a condition is met means that the probability of the subject to beresponsive to a particular treatment is higher when the condition is metthan when the condition is not met. The probability to be responsive toa particular treatment can be higher by, for example, 5%, 10%, 25%, 50%,100%, 200%, or more in a subject who meets a particular conditioncompared to a subject who does not meet the condition. For example, acancer patient is “likely” to be responsive to an FTI treatment when thesubject is a carrier of an HRAS mutation means that the probability of asubject to be responsive to FTI treatment is 5%, 10%, 25%, 50%, 100%,200%, or more higher in a subject who is a carrier of an HRAS mutationcompared to a subject who is not a carrier of an HRAS mutation.

Ras proteins are GTPases that regulate proliferation and by transducingbiological information from extracellular signals to the nucleus.Mammalian cells express three ras genes that encode four Ras proteins,which are HRAS, N-Ras, KA-Ras and KB-Ras. KA-Ras and KB-Ras are alsogenerally referred to as K-Ras. Ras proteins exist in either an active,GTP-bound or an inactive, GDP-bound, state. Mutant RAS proteinsaccumulate in the GTP-bound conformation due to defective intrinsicGTPase activity and/or resistance to inactivation by GTPase activatingproteins (GAPs). Constitutive RAS signaling is mediated by mutationsthat prevent GTP hydrolysis, locking RAS in a permanently active state.In addition, RAS GTPases require lipid post-translational modificationin the form of farnesylation or geranylgeranylation for their malignanttransforming activity. Of the three RAS species (HRAS, KRAS, NRAS), HRASis unique in the fact that it can be farnesylated but notgeranylgeranylated. Consequently, farnesyl transferase inhibitors (FTIs)have been shown to inhibit the farnesylation of HRAS, prevent itsassociation with the plasma membrane, inhibit downstream signaltransduction pathways and inhibit tumor growth (Reviewed by Berndt etal. Nature Reviews Cancer 11:775-91). The Q22K HRAS mutation has beenobserved in Costello syndrome (Sheffield et al. Ped Dev Pathology 18,237-244, 2015), a developmental and tumor predisposition disorder causedby germline HRAS mutation, and while its ability to drive neoplastictransformation has not been established, this mutation is in a highlyconserved region across RAS proteins and Q22K KRAS has been establishedas a driver mutation (Tsukuda et al. Biochem Biophys Res Commun 2000;278:653-58).

An exemplary amino acid sequence and a corresponding encoding nucleicacid sequence of human HRAS (GENBANK: CR536579.1 GI:49168641) areprovided below:

(SEQ ID NO: 1) MTEYKLVVVG AGGVGKSALT IQLIQNHFVDEYDPTIEDSY RKQVVIDGET CLLDILDTAG QEEYSAMRDQ YMRTGEGFLC VFAINNTKSFEDIHQYREQI KRVKDSDDVP MVLVGNKCDL AARTVESRQA QDLARSYGIP YIETSAKTRQGVEDAFYTLV REIRQHKLRK LNPPDESGPG CMSCKCVLS (SEQ ID NO: 2)ATGACGGAAT ATAAGCTGGT GGTGGTGGGC GCCGGCGGTG TGGGCAAGAG TGCGCTGACCATCCAGCTGA TCCAGAACCA CTTTGTGGAC GAATACGACC CCACTATAGA GGATTCCTACCGGAAGCAGG TGGTCATTGA TGGGGAGACG TGCCTGTTGG ACATCCTGGA TACCGCCGGCCAGGAGGAGT ACAGCGCCAT GCGGGACCAG TACATGCGCA CCGGGGAGGG CTTCCTGTGTGTGTTTGCCA TCAACAACAC CAAGTCTTTT GAGGACATCC ACCAGTACAG GGAGCAGATCAAACGGGTGA AGGACTCGGA TGACGTGCCC ATGGTGCTGG TGGGGAACAA GTGTGACCTGGCTGCACGCA CTGTGGAATC TCGGCAGGCT CAGGACCTCG CCCGAAGCTA CGGCATCCCCTACATCGAGA CCTCGGCCAA GACCCGGCAG GGAGTGGAGG ATGCCTTCTA CACGTTGGTGCGTGAGATCC GGCAGCACAA GCTGCGGAAG CTGAACCCTC CTGATGAGAG TGGCCCCGGCTGCATGAGCT GCAAGTGTGT GCTCTCCTGA.

Ras isoforms are farnesylated. Farnesyltransferase (FTase) have crucialroles in the post-translational modifications of Ras proteins. A way ofinterfering with Ras function is the inhibition of FTase, the enzymecoupling a 15-carbon isoprenyl group to Ras proteins, byFarnesyltransferase Inhibitors (“FTI”). FTIs are a class of biologicallyactive anticancer drugs that inhibit farnesylation of a wide range oftarget proteins, including Ras. The FTIs block Ras activation throughinhibition of FTase, ultimately resulting in cell growth arrest. Thus,it was predicted that FTIs would be effective therapeutic agents in thetreatment of cancer.

Thirty percent of all human cancers express oncogenically activated Ras.The high prevalence of mutated Ras, found in 30% of all human cancers,makes this pathway an attractive target for anticancer drug development.Initially, it was predicted that the Ras mutation(s) that led toconstitutively active RAS pathway can serve as a biomarker for patientresponse to FTIs, which was based on the preclinical evidence that FTIscould block RAS-transformed cells. (Raponi et al., Blood 111:2589-96(2008)).

As used herein, the term “HRAS mutation” refers to an activationmutation in an HRAS gene or HRAS protein. An HRAS mutation can refer toeither a genetic alternation in the DNA sequence of the HRAS gene thatresults in activation of the corresponding HRAS protein, or thealteration in the amino acid sequence of an HRAS protein that results inits activation. Thus, the term “HRAS mutation” as used herein does notinclude an alternation in a HRAS gene that does not result in theactivation of the HRAS protein, or an alternation of a HRAS proteinsequence that does not lead to its activation. Accordingly, a sample ora subject that does not have any “HRAS mutation” as used herein canstill have a mutation in the HRAS gene that does not affect the activityof the HRAS protein or a mutation that impairs the activity of the HRASprotein, or have a mutation in an HRAS protein that does not affect itsactivity or a mutation that impairs its activity. A sample or a subjectcan have multiple copies of the HRAS gene. A sample or a subject canalso have both wild type and mutant HRAS proteins. As used herein, asample or a subject having an HRAS mutation can also have a copy of wildtype HRAS gene and/or the wild type HRAS protein. A sample or a subjectthat is determined to “have wild type HRAS,” as used herein, refers tothe sample or subject that only has the wild type HRAS gene and the wildtype HRAS protein, and no HRAS mutation.

In some embodiments, the HRAS mutation can include at least one mutationat a codon selected from the group consisting of G12, G13, Q61, Q22,K117, and A146.

2. Farnesyltransferase Inhibitors for Cancer Treatment 2.1.Farnesyltransferase Inhibitors

Provided herein are methods to treat squamous cell carcinoma of the headand neck with an FTI in a selected cancer patient or a selectedpopulation of cancer patients. The representative FTIs roughly belong totwo classes (Shen et al., Drug Disc. Today 20:2 (2015)). The FTIs in thefirst class have the basic framework of farnesyldiphosphate (FPP). Forinstance, FPP analogs with a malonic acid group (Ta) were reported to beFTIs that compete with FPP (Duez, S. et al. Bioorg. Med. Chem.18:543-556 (2010)). In addition, imidazole-containing derivatives linkedby an acidic substituent and a peptidyl chain were also synthesized asbisubstrate FTIs, and the designed bisubstrate inhibitors have betteraffinities than FPP. The FTIs in the second class are peptidomimeticmolecules, which can be divided into two groups, namely thiol andnon-thiol FTIs. Regarding the thiol FTIs, for instance L-739749, aselective peptidomimetic FTI shows potent antitumor activity in nudemice without system toxicity (Kohl, N. E. et al. PNAS 91:9141-9145(1994)). Additionally, a variety of thiol inhibitors were alsodeveloped, such as tripeptidyl FTIs (Lee, H-Y. et al. Bioorg. Med. Chem.Lett. 12:1599-1602 (2002)).

For non-thiol FTIs, the heterocycles were therefore widely used tosubstitute the thiol group to contact with the zinc ion in the bindingsite. According to the structures of pharmacophoric groups, the nonthiolFTIs can be divided into three classes. The first class is featured bydifferent monocyclic rings, such as L-778123, an FTI in Phase I clinicaltrials for solid tumors and lymphoma. L-778123 binds into the CAAXpeptide site and competes with the CAAX substrate offarnesyltransferase. The second class is represented by tipifarnib inPhase III trials and BMS-214662 in Phase III trials, which are composedof diverse monocyclic rings and bicyclic rings (Harousseau et al. Blood114:1166-1173 (2009)). The representative inhibitor of the third classis lonafarnib, which is active in Ras-dependent and -independentmalignant tumors, and has entered Phase III clinical trials forcombating carcinoma, leukemia, and myelodysplastic syndrome. Lonafarnibis an FTI with a tricycle core, which contains a central seven-memberedring fused with two six-membered aromatic rings.

Thus, FTIs as described herein can take on a multitude of forms butshare the essential inhibitory function of interfering with or lesseningthe farnesylation of proteins implicated in cancer and proliferativediseases.

Numerous FTIs are within the scope of this disclosure and include thosedescribed in U.S. Pat. Nos. 5,976,851; 5,972,984; 5,972,966; 5,968,965;5,968,952; 6,187,786; 6,169,096; 6,037,350; 6,177,432; 5,965,578;5,965,539; 5,958,939; 5,939,557; 5,936,097; 5,891,889; 5,889,053;5,880,140; 5,872,135; 5,869,682; 5,861,529; 5,859,015; 5,856,439;5,856,326; 5,852,010; 5,843,941; 5,807,852; 5,780,492; 5,773,455;5,767,274; 5,756,528; 5,750,567; 5,721,236; 5,700,806; 5,661,161;5,602,098; 5,585,359; 5,578,629; 5,534,537; 5,532,359; 5,523,430;5,504,212; 5,491,164; 5,420,245; and 5,238,922, the disclosures of whichare hereby incorporated by reference in their entireties.

FTIs within the scope of this disclosure also include those described inThomas et al., Biologics 1: 415-424 (2007); Shen et al., Drug Disc.Today 20:2 (2015); Appels et al., The Oncologist 10:565-578 (2005), thedisclosures of which are hereby incorporated by reference in theirentireties.

In some embodiments, the FTIs include Arglabin(i.e.l(R)-10-epoxy-5(S),7(S)-guaia-3(4),11(13)-dien-6,12-olide describedin WO-98/28303 (NuOncology Labs); perrilyl alcohol described inWO-99/45912 (Wisconsin Genetics); SCH-66336 (lonafarnib), i.e.(+)-(R)-4-[2-[4-(3,10-dibromo-8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl)piperidin-1-yl]-2-oxoethyl]piperidine-1-carboxamide,described in U.S. Pat. No. 5,874,442 (Schering); L778123, i.e.1-(3-chlorophenyl)-4-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-2-piperazinone,described in WO-00/01691 (Merck); L739749, i.e. compound2(S)-[2(S)-[2(R)-amino-3-mercapto]propylamino-3(S)-methyl]-pentyloxy-3-phenylpropionyl-methioninesulfone described in WO-94/10138 (Merck); FTI-277, i.e., methyl{N-[2-phenyl-4-N [2(R)-amino-3-mecaptopropylamino] benzoyl]}-methionate(Calbiochem); L744832, i.e, 2S)-2-[[(2S)-2-[(2S,3S)-2-[(2R)-2-amino-3-mercaptopropyl]amino]-3-methylpentyl]oxy]-1-oxo-3-phenylpropyl]amino]-4-(methylsulfonyl)-butanoicacid 1-methylethyl ester (Biomol International L.P.); CP-609,754(Pfizer), i.e.,(R)-6-[(4-chlorophenyl)-hydroxyl-(1-methyl-1-H-imidazol-5-yl)-methyl]-4-(3-ethynylphenyl)-1-methyl-2-(1H)-quinonlinoneand(R)-6-[(4-chlorophenyl)-hydroxyl-(3-methyl-3-H-imidazol-4-yl)-methyl]-4-(3-ethynylphenyl)-1-methyl-2-(1H)-quinolinone;R208176 (Johnson & Johnson), i.e., JNJ-17305457, or(R)-1-(4-chlorophenyl)-1-[5-(3-chlorophenyl)tetrazolo[1,5-a]quinazolin-7-yl]-1-(1-methyl-1H-imidazol-5-yl)methanamine;AZD3409 (AstraZeneca), i.e. (S)-isopropyl2-(2-(4-fluorophenethyl)-5-((((2S,4S)-4-(nicotinoylthio)pyrrolidin-2-yl)methyl)amino)benzamido)-4-(methylthio)butanoate;BMS 214662 (Bristol-Myers Squibb), i.e.(R)-2,3,4,5-tetrahydro-1-(IH-imidazol-4-ylmethyl)-3-(phenylmethyl)-4-(2-thienylsulphonyl)-1H-1,4-benzodiazapine-7-carbonitrile,described in WO 97/30992 (Bristol Myers Squibb) and Pfizer compounds (A)and (B) described in WO-00/12498 and WO-00/12499.

In some embodiments, the FTI are the non-peptidal, so-called “smallmolecule” therapeutics, such as are quinolines or quinoline derivativesincluding:

-   7-(3-chlorophenyl)-9-[(4-chlorophenyl)-1H-imidazol-1-ylmethyl]-2,3-dihydro-o-1H,5H-benzo[ij]quinolizin-5-one,-   7-(3-chlorophenyl)-9-[(4-chlorophenyl)-1H-imidazol-1-ylmethyl]-1,2-dihydro-o-4H-pyrrolo[3,2,1-ij]quinoline-4-one,-   8-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl),    methyl]-6-(3-chloroph-enyl)-1,2-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-4-one,    and-   8-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-6-(3-chlorophe-nyl)-2,3-dihydro-1H,5H-benzo[ij]quinolizin-5-one.

Tipifarnib is a nonpeptidomimetic FTI (Thomas et al., Biologics 1:415-424 (2007)). It is a 4,6-disubstituted-1-methylquinolin-2-onederivative((B)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone))that was obtained by optimization of a quinolone lead identified fromcompound library screening. Tipifarnib competitively inhibits the CAAXpeptide binding site of FTase and is an extremely potent and highlyselective inhibitor of farnesylation. Tipifarnib has manageable safetyprofile as single agent therapy and is reasonably well tolerated in man.

Tipifarnib is synthesized by the condensation of the anion of1-methylimidazole with a 6-(4-chlorobenzoyl) quinolone derivative,followed by dehydration. The quinolone intermediate was prepared in foursteps by cyclization of N-phenyl-3-(3-chlorophenyl)-2-propenamide,acylation, oxidation and N-methylation. Tipifarnib is a potent inhibitorof FTase in vitro and is orally active in a variety of animal models.

In some embodiments, provided herein is a method of treating cancer in asubject with an FTI or a pharmaceutical composition having FTI, orselecting a cancer patient for an FTI treatment. The pharmaceuticalcompositions provided herein contain therapeutically effective amountsof an FTI and a pharmaceutically acceptable carrier, diluent orexcipient. In some embodiments, the FTI is tipifarnib; arglabin;perrilyl alcohol; lonafarnib (SCH-66336); L778123; L739749; FTI-277;L744832; 8208176; BMS 214662; AZD3409; or CP-609,754. In someembodiments, the FTI is tipifarnib.

2.2. Formulations

The FTI can be formulated into suitable pharmaceutical preparations suchas solutions, suspensions, tablets, dispersible tablets, pills,capsules, powders, sustained release formulations or elixirs, for oraladministration or in sterile solutions or suspensions for ophthalmic orparenteral administration, as well as transdermal patch preparation anddry powder inhalers. Typically the FTI is formulated into pharmaceuticalcompositions using techniques and procedures well known in the art (see,e.g., Ansel Introduction to Pharmaceutical Dosage Forms, Seventh Edition1999).

In the compositions, effective concentrations of the FTI andpharmaceutically acceptable salts is (are) mixed with a suitablepharmaceutical carrier or vehicle. In certain embodiments, theconcentrations of the FTI in the compositions are effective for deliveryof an amount, upon administration, that treats, prevents, or amelioratesone or more of the symptoms and/or progression of cancer, includinghaematological cancers and solid tumors.

The compositions can be formulated for single dosage administration. Toformulate a composition, the weight fraction of the FTI is dissolved,suspended, dispersed or otherwise mixed in a selected vehicle at aneffective concentration such that the treated condition is relieved orameliorated. Pharmaceutical carriers or vehicles suitable foradministration of the FTI provided herein include any such carriersknown to those skilled in the art to be suitable for the particular modeof administration.

In addition, the FTI can be formulated as the sole pharmaceuticallyactive ingredient in the composition or may be combined with otheractive ingredients. Liposomal suspensions, including tissue-targetedliposomes, such as tumor-targeted liposomes, may also be suitable aspharmaceutically acceptable carriers. These may be prepared according tomethods known to those skilled in the art. For example, liposomeformulations may be prepared as known in the art. Briefly, liposomessuch as multilamellar vesicles (MLV's) may be formed by drying down eggphosphatidyl choline and brain phosphatidyl serine (7:3 molar ratio) onthe inside of a flask. A solution of an FTI provided herein in phosphatebuffered saline lacking divalent cations (PBS) is added and the flaskshaken until the lipid film is dispersed. The resulting vesicles arewashed to remove unencapsulated compound, pelleted by centrifugation,and then resuspended in PBS.

The FTI is included in the pharmaceutically acceptable carrier in anamount sufficient to exert a therapeutically useful effect in theabsence of undesirable side effects on the patient treated. Thetherapeutically effective concentration may be determined empirically bytesting the compounds in in vitro and in vivo systems described hereinand then extrapolated therefrom for dosages for humans.

The concentration of FTI in the pharmaceutical composition will dependon absorption, tissue distribution, inactivation and excretion rates ofthe FTI, the physicochemical characteristics of the FTI, the dosageschedule, and amount administered as well as other factors known tothose of skill in the art. For example, the amount that is delivered issufficient to ameliorate one or more of the symptoms of cancer,including hematopoietic cancers and solid tumors.

In certain embodiments, a therapeutically effective dosage shouldproduce a serum concentration of active ingredient from about 0.1 ng/mlto about 50-100 μg/ml. In one embodiment, the pharmaceuticalcompositions provide a dosage of from about 0.001 mg to about 2000 mg ofcompound per kilogram of body weight per day. Pharmaceutical dosage unitforms are prepared to provide from about 1 mg to about 1000 mg and incertain embodiments, from about 10 to about 500 mg of the essentialactive ingredient or a combination of essential ingredients per dosageunit form.

The FTI may be administered at once, or may be divided into a number ofsmaller doses to be administered at intervals of time. It is understoodthat the precise dosage and duration of treatment is a function of thedisease being treated and may be determined empirically using knowntesting protocols or by extrapolation from in vivo or in vitro testdata. It is to be noted that concentrations and dosage values may alsovary with the severity of the condition to be alleviated. It is to befurther understood that for any particular subject, specific dosageregimens should be adjusted over time according to the individual needand the professional judgment of the person administering or supervisingthe administration of the compositions, and that the concentrationranges set forth herein are exemplary only and are not intended to limitthe scope or practice of the claimed compositions.

Thus, effective concentrations or amounts of one or more of thecompounds described herein or pharmaceutically acceptable salts thereofare mixed with a suitable pharmaceutical carrier or vehicle forsystemic, topical or local administration to form pharmaceuticalcompositions. Compounds are included in an amount effective forameliorating one or more symptoms of, or for treating, retardingprogression, or preventing. The concentration of active compound in thecomposition will depend on absorption, tissue distribution,inactivation, excretion rates of the active compound, the dosageschedule, amount administered, particular formulation as well as otherfactors known to those of skill in the art.

The compositions are intended to be administered by a suitable route,including but not limited to orally, parenterally, rectally, topicallyand locally. For oral administration, capsules, tablets, suspensions,and solutions can be formulated. The compositions are in liquid,semi-liquid or solid form and are formulated in a manner suitable foreach route of administration.

Solutions or suspensions used for parenteral, intradermal, subcutaneous,or topical application can include any of the following components: asterile diluent, such as water for injection, saline solution, fixedoil, polyethylene glycol, glycerine, propylene glycol, dimethylacetamide or other synthetic solvent; antimicrobial agents, such asbenzyl alcohol and methyl parabens; antioxidants, such as ascorbic acidand sodium bisulfate; chelating agents, such asethylenediaminetetraacetic acid (EDTA); buffers, such as acetates,citrates and phosphates; and agents for the adjustment of tonicity suchas sodium chloride or dextrose. Parenteral preparations can be enclosedin ampules, pens, disposable syringes or single or multiple dose vialsmade of glass, plastic or other suitable material.

In instances in which the FTI exhibits insufficient solubility, methodsfor solubilizing compounds can be used. Such methods are known to thoseof skill in this art, and include, but are not limited to, usingcosolvents, such as dimethylsulfoxide (DMSO), using surfactants, such asTWEEN®, or dissolution in aqueous sodium bicarbonate.

Upon mixing or addition of the compound(s), the resulting mixture may bea solution, suspension, emulsion or the like. The form of the resultingmixture depends upon a number of factors, including the intended mode ofadministration and the solubility of the compound in the selectedcarrier or vehicle. The effective concentration is sufficient forameliorating the symptoms of the disease, disorder or condition treatedand may be empirically determined.

The pharmaceutical compositions are provided for administration tohumans and animals in unit dosage forms, such as tablets, capsules,pills, powders, granules, sterile parenteral solutions or suspensions,and oral solutions or suspensions, and oil water emulsions containingsuitable quantities of the compounds or pharmaceutically acceptablesalts thereof. The pharmaceutically therapeutically active compounds andsalts thereof are formulated and administered in unit dosage forms ormultiple dosage forms. Unit dose forms as used herein refer tophysically discrete units suitable for human and animal subjects andpackaged individually as is known in the art. Each unit dose contains apredetermined quantity of the therapeutically active compound sufficientto produce the desired therapeutic effect, in association with therequired pharmaceutical carrier, vehicle or diluent. Examples of unitdose forms include ampules and syringes and individually packagedtablets or capsules. Unit dose forms may be administered in fractions ormultiples thereof. A multiple dose form is a plurality of identical unitdosage forms packaged in a single container to be administered insegregated unit dose form. Examples of multiple dose forms includevials, bottles of tablets or capsules or bottles of pints or gallons.Hence, multiple dose form is a multiple of unit doses which are notsegregated in packaging.

Sustained-release preparations can also be prepared. Suitable examplesof sustained-release preparations include semipermeable matrices ofsolid hydrophobic polymers containing the compound provided herein,which matrices are in the form of shaped articles, e.g., films, ormicrocapsule. Examples of sustained-release matrices includeiontophoresis patches, polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides,copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated compound remain in the body for a long time,they may denature or aggregate as a result of exposure to moisture at37° C., resulting in a loss of biological activity and possible changesin their structure. Rational strategies can be devised for stabilizationdepending on the mechanism of action involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Dosage forms or compositions containing active ingredient in the rangeof 0.005% to 100% with the balance made up from non toxic carrier may beprepared. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by the incorporation of any of the normallyemployed excipients, such as, for example pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, talcum, cellulosederivatives, sodium crosscarmellose, glucose, sucrose, magnesiumcarbonate or sodium saccharin. Such compositions include solutions,suspensions, tablets, capsules, powders and sustained releaseformulations, such as, but not limited to, implants andmicroencapsulated delivery systems, and biodegradable, biocompatiblepolymers, such as collagen, ethylene vinyl acetate, polyanhydrides,polyglycolic acid, polyorthoesters, polylactic acid and others. Methodsfor preparation of these compositions are known to those skilled in theart. The contemplated compositions may contain about 0.001% 100% activeingredient, in certain embodiments, about 0.1-85% or about 75-95%.

The FTI or pharmaceutically acceptable salts can be prepared withcarriers that protect the compound against rapid elimination from thebody, such as time release formulations or coatings.

The compositions can include other active compounds to obtain desiredcombinations of properties. The compounds provided herein, orpharmaceutically acceptable salts thereof as described herein, can alsobe administered together with another pharmacological agent known in thegeneral art to be of value in treating one or more of the diseases ormedical conditions referred to hereinabove, such as diseases related tooxidative stress.

Lactose-free compositions provided herein can contain excipients thatare well known in the art and are listed, for example, in the U.S.Pharmocopia (USP) SP (XXI)/NF (XVI). In general, lactose-freecompositions contain an active ingredient, a binder/filler, and alubricant in pharmaceutically compatible and pharmaceutically acceptableamounts. Exemplary lactose-free dosage forms contain an activeingredient, microcrystalline cellulose, pre-gelatinized starch andmagnesium stearate.

Further encompassed are anhydrous pharmaceutical compositions and dosageforms containing a compound provided herein. For example, the additionof water (e.g., 5%) is widely accepted in the pharmaceutical arts as ameans of simulating long-term storage in order to determinecharacteristics such as shelf-life or the stability of formulations overtime. See, e.g., Jens T. Carstensen, Drug Stability: Principles &Practice, 2d. Ed., Marcel Dekker, NY, NY, 1995, pp. 379-80. In effect,water and heat accelerate the decomposition of some compounds. Thus, theeffect of water on a formulation can be of great significance sincemoisture and/or humidity are commonly encountered during manufacture,handling, packaging, storage, shipment and use of formulations.

Anhydrous pharmaceutical compositions and dosage forms provided hereincan be prepared using anhydrous or low moisture containing ingredientsand low moisture or low humidity conditions. Pharmaceutical compositionsand dosage forms that comprise lactose and at least one activeingredient that comprises a primary or secondary amine are anhydrous ifsubstantial contact with moisture and/or humidity during manufacturing,packaging, and/or storage is expected.

An anhydrous pharmaceutical composition should be prepared and storedsuch that its anhydrous nature is maintained. Accordingly, anhydrouscompositions are packaged using materials known to prevent exposure towater such that they can be included in suitable formulary kits.Examples of suitable packaging include, but are not limited to,hermetically sealed foils, plastics, unit dose containers (e.g., vials),blister packs and strip packs.

Oral pharmaceutical dosage forms are either solid, gel or liquid. Thesolid dosage forms are tablets, capsules, granules, and bulk powders.Types of oral tablets include compressed, chewable lozenges and tabletswhich may be enteric coated, sugar coated or film coated. Capsules maybe hard or soft gelatin capsules, while granules and powders may beprovided in non effervescent or effervescent form with the combinationof other ingredients known to those skilled in the art.

In certain embodiments, the formulations are solid dosage forms, such ascapsules or tablets. The tablets, pills, capsules, troches and the likecan contain any of the following ingredients, or compounds of a similarnature: a binder; a diluent; a disintegrating agent; a lubricant; aglidant; a sweetening agent; and a flavoring agent.

Examples of binders include microcrystalline cellulose, gum tragacanth,glucose solution, acacia mucilage, gelatin solution, sucrose and starchpaste. Lubricants include talc, starch, magnesium or calcium stearate,lycopodium and stearic acid. Diluents include, for example, lactose,sucrose, starch, kaolin, salt, mannitol and dicalcium phosphate.Glidants include, but are not limited to, colloidal silicon dioxide.Disintegrating agents include crosscarmellose sodium, sodium starchglycolate, alginic acid, corn starch, potato starch, bentonite,methylcellulose, agar and carboxymethylcellulose. Coloring agentsinclude, for example, any of the approved certified water soluble FD andC dyes, mixtures thereof; and water insoluble FD and C dyes suspended onalumina hydrate. Sweetening agents include sucrose, lactose, mannitoland artificial sweetening agents such as saccharin, and any number ofspray dried flavors. Flavoring agents include natural flavors extractedfrom plants such as fruits and synthetic blends of compounds whichproduce a pleasant sensation, such as, but not limited to peppermint andmethyl salicylate. Wetting agents include propylene glycol monostearate,sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylenelaural ether. Emetic coatings include fatty acids, fats, waxes, shellac,ammoniated shellac and cellulose acetate phthalates. Film coatingsinclude hydroxyethylcellulose, sodium carboxymethylcellulose,polyethylene glycol 4000 and cellulose acetate phthalate.

When the dosage unit form is a capsule, it can contain, in addition tomaterial of the above type, a liquid carrier such as a fatty oil. Inaddition, dosage unit forms can contain various other materials whichmodify the physical form of the dosage unit, for example, coatings ofsugar and other enteric agents. The compounds can also be administeredas a component of an elixir, suspension, syrup, wafer, sprinkle, chewinggum or the like. A syrup may contain, in addition to the activecompounds, sucrose as a sweetening agent and certain preservatives, dyesand colorings and flavors.

Pharmaceutically acceptable carriers included in tablets are binders,lubricants, diluents, disintegrating agents, coloring agents, flavoringagents, and wetting agents. Enteric coated tablets, because of theenteric coating, resist the action of stomach acid and dissolve ordisintegrate in the neutral or alkaline intestines. Sugar coated tabletsare compressed tablets to which different layers of pharmaceuticallyacceptable substances are applied. Film coated tablets are compressedtablets which have been coated with a polymer or other suitable coating.Multiple compressed tablets are compressed tablets made by more than onecompression cycle utilizing the pharmaceutically acceptable substancespreviously mentioned. Coloring agents may also be used in the abovedosage forms. Flavoring and sweetening agents are used in compressedtablets, sugar coated, multiple compressed and chewable tablets.Flavoring and sweetening agents are especially useful in the formationof chewable tablets and lozenges.

Liquid oral dosage forms include aqueous solutions, emulsions,suspensions, solutions and/or suspensions reconstituted from noneffervescent granules and effervescent preparations reconstituted fromeffervescent granules. Aqueous solutions include, for example, elixirsand syrups. Emulsions are either oil in-water or water in oil.

Elixirs are clear, sweetened, hydroalcoholic preparations.Pharmaceutically acceptable carriers used in elixirs include solvents.Syrups are concentrated aqueous solutions of a sugar, for example,sucrose, and may contain a preservative. An emulsion is a two phasesystem in which one liquid is dispersed in the form of small globulesthroughout another liquid. Pharmaceutically acceptable carriers used inemulsions are non aqueous liquids, emulsifying agents and preservatives.Suspensions use pharmaceutically acceptable suspending agents andpreservatives. Pharmaceutically acceptable substances used in noneffervescent granules, to be reconstituted into a liquid oral dosageform, include diluents, sweeteners and wetting agents. Pharmaceuticallyacceptable substances used in effervescent granules, to be reconstitutedinto a liquid oral dosage form, include organic acids and a source ofcarbon dioxide. Coloring and flavoring agents are used in all of theabove dosage forms.

Solvents include glycerin, sorbitol, ethyl alcohol and syrup. Examplesof preservatives include glycerin, methyl and propylparaben, benzoicadd, sodium benzoate and alcohol. Examples of non aqueous liquidsutilized in emulsions include mineral oil and cottonseed oil. Examplesof emulsifying agents include gelatin, acacia, tragacanth, bentonite,and surfactants such as polyoxyethylene sorbitan monooleate. Suspendingagents include sodium carboxymethylcellulose, pectin, tragacanth, Veegumand acacia. Diluents include lactose and sucrose. Sweetening agentsinclude sucrose, syrups, glycerin and artificial sweetening agents suchas saccharin. Wetting agents include propylene glycol monostearate,sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylenelauryl ether. Organic adds include citric and tartaric acid. Sources ofcarbon dioxide include sodium bicarbonate and sodium carbonate. Coloringagents include any of the approved certified water soluble FD and Cdyes, and mixtures thereof. Flavoring agents include natural flavorsextracted from plants such fruits, and synthetic blends of compoundswhich produce a pleasant taste sensation.

For a solid dosage form, the solution or suspension, in for examplepropylene carbonate, vegetable oils or triglycerides, is encapsulated ina gelatin capsule. Such solutions, and the preparation and encapsulationthereof, are disclosed in U.S. Pat. Nos. 4,328,245; 4,409,239; and4,410,545. For a liquid dosage form, the solution, e.g., for example, ina polyethylene glycol, may be diluted with a sufficient quantity of apharmaceutically acceptable liquid carrier, e.g., water, to be easilymeasured for administration.

Alternatively, liquid or semi solid oral formulations may be prepared bydissolving or dispersing the active compound or salt in vegetable oils,glycols, triglycerides, propylene glycol esters (e.g., propylenecarbonate) and other such carriers, and encapsulating these solutions orsuspensions in hard or soft gelatin capsule shells. Other usefulformulations include, but are not limited to, those containing acompound provided herein, a dialkylated mono- or poly-alkylene glycol,including, but not limited to, 1,2-dimethoxymethane, diglyme, triglyme,tetraglyme, polyethylene glycol-350-dimethyl ether, polyethyleneglycol-550-dimethyl ether, polyethylene glycol-750-dimethyl etherwherein 350, 550 and 750 refer to the approximate average molecularweight of the polyethylene glycol, and one or more antioxidants, such asbutylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propylgallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine,lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoricacid, thiodipropionic acid and its esters, and dithiocarbamates.

Other formulations include, but are not limited to, aqueous alcoholicsolutions including a pharmaceutically acceptable acetal. Alcohols usedin these formulations are any pharmaceutically acceptable water-misciblesolvents having one or more hydroxyl groups, including, but not limitedto, propylene glycol and ethanol. Acetals include, but are not limitedto, di(lower alkyl) acetals of lower alkyl aldehydes such asacetaldehyde diethyl acetal.

In all embodiments, tablets and capsules formulations may be coated asknown by those of skill in the art in order to modify or sustaindissolution of the active ingredient. Thus, for example, they may becoated with a conventional enterically digestible coating, such asphenylsalicylate, waxes and cellulose acetate phthalate.

Parenteral administration, generally characterized by injection, eithersubcutaneously, intramuscularly or intravenously is also providedherein. Injectables can be prepared in conventional forms, either asliquid solutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Suitableexcipients are, for example, water, saline, dextrose, glycerol orethanol. In addition, if desired, the pharmaceutical compositions to beadministered may also contain minor amounts of non toxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,stabilizers, solubility enhancers, and other such agents, such as forexample, sodium acetate, sorbitan monolaurate, triethanolamine oleateand cyclodextrins. Implantation of a slow release or sustained releasesystem, such that a constant level of dosage is maintained is alsocontemplated herein. Briefly, a compound provided herein is dispersed ina solid inner matrix, e.g., polymethylmethacrylate,polybutylmethacrylate, plasticized or unplasticized polyvinylchloride,plasticized nylon, plasticized polyethyleneterephthalate, naturalrubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene,ethylene-vinylacetate copolymers, silicone rubbers,polydimethylsiloxanes, silicone carbonate copolymers, hydrophilicpolymers such as hydrogels of esters of acrylic and methacrylic acid,collagen, cross-linked polyvinylalcohol and cross-linked partiallyhydrolyzed polyvinyl acetate, that is surrounded by an outer polymericmembrane, e.g., polyethylene, polypropylene, ethylene/propylenecopolymers, ethylene/ethyl acrylate copolymers, ethylene/vinylacetatecopolymers, silicone rubbers, polydimethyl siloxanes, neoprene rubber,chlorinated polyethylene, polyvinylchloride, vinylchloride copolymerswith vinyl acetate, vinylidene chloride, ethylene and propylene, ionomerpolyethylene terephthalate, butyl rubber epichlorohydrin rubbers,ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcoholterpolymer, and ethylene/vinyloxyethanol copolymer, that is insoluble inbody fluids. The compound diffuses through the outer polymeric membranein a release rate controlling step. The percentage of active compoundcontained in such parenteral compositions is highly dependent on thespecific nature thereof, as well as the activity of the compound and theneeds of the subject.

Parenteral administration of the compositions includes intravenous,subcutaneous and intramuscular administrations. Preparations forparenteral administration include sterile solutions ready for injection,sterile dry soluble products, such as lyophilized powders, ready to becombined with a solvent just prior to use, including hypodermic tablets,sterile suspensions ready for injection, sterile dry insoluble productsready to be combined with a vehicle just prior to use and sterileemulsions. The solutions may be either aqueous or nonaqueous.

If administered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropylene glycol and mixtures thereof.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances.

Examples of aqueous vehicles include Sodium Chloride Injection, RingersInjection, Isotonic Dextrose Injection, Sterile Water Injection,Dextrose and Lactated Ringers Injection. Nonaqueous parenteral vehiclesinclude fixed oils of vegetable origin, cottonseed oil, corn oil, sesameoil and peanut oil. Antimicrobial agents in bacteriostatic orfungistatic concentrations must be added to parenteral preparationspackaged in multiple dose containers which include phenols or cresols,mercurials, benzyl alcohol, chlorobutanol, methyl and propyl phydroxybenzoic acid esters, thimerosal, benzalkonium chloride andbenzethonium chloride. Isotonic agents include sodium chloride anddextrose. Buffers include phosphate and citrate. Antioxidants includesodium bisulfate. Local anesthetics include procaine hydrochloride.Suspending and dispersing agents include sodium carboxymethylcelluose,hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifyingagents include Polysorbate 80 (TWEEN® 80). A sequestering or chelatingagent of metal ions include EDTA. Pharmaceutical carriers also includeethyl alcohol, polyethylene glycol and propylene glycol for watermiscible vehicles and sodium hydroxide, hydrochloric acid, citric acidor lactic acid for pH adjustment.

The concentration of the FTI is adjusted so that an injection providesan effective amount to produce the desired pharmacological effect. Theexact dose depends on the age, weight and condition of the patient oranimal as is known in the art. The unit dose parenteral preparations arepackaged in an ampule, a vial or a syringe with a needle. Allpreparations for parenteral administration must be sterile, as is knownand practiced in the art.

Illustratively, intravenous or intraarterial infusion of a sterileaqueous solution containing an FTI is an effective mode ofadministration. Another embodiment is a sterile aqueous or oily solutionor suspension containing an active material injected as necessary toproduce the desired pharmacological effect.

Injectables are designed for local and systemic administration.Typically a therapeutically effective dosage is formulated to contain aconcentration of at least about 0.1% w/w up to about 90% w/w or more,such as more than 1% w/w of the active compound to the treatedtissue(s). The active ingredient may be administered at once, or may bedivided into a number of smaller doses to be administered at intervalsof time. It is understood that the precise dosage and duration oftreatment is a function of the tissue being treated and may bedetermined empirically using known testing protocols or by extrapolationfrom in vivo or in vitro test data. It is to be noted thatconcentrations and dosage values may also vary with the age of theindividual treated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of theformulations, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed formulations.

The FTI can be suspended in micronized or other suitable form or may bederivatized to produce a more soluble active product or to produce aprodrug. The form of the resulting mixture depends upon a number offactors, including the intended mode of administration and thesolubility of the compound in the selected carrier or vehicle. Theeffective concentration is sufficient for ameliorating the symptoms ofthe condition and may be empirically determined.

Of interest herein are also lyophilized powders, which can bereconstituted for administration as solutions, emulsions and othermixtures. They can also be reconstituted and formulated as solids orgels.

The sterile, lyophilized powder is prepared by dissolving an FTIprovided herein, or a pharmaceutically acceptable salt thereof, in asuitable solvent. The solvent may contain an excipient which improvesthe stability or other pharmacological component of the powder orreconstituted solution, prepared from the powder. Excipients that may beused include, but are not limited to, dextrose, sorbital, fructose, cornsyrup, xylitol, glycerin, glucose, sucrose or other suitable agent. Thesolvent may also contain a buffer, such as citrate, sodium or potassiumphosphate or other such buffer known to those of skill in the art at, inone embodiment, about neutral pH. Subsequent sterile filtration of thesolution followed by lyophilization under standard conditions known tothose of skill in the art provides the desired formulation. Generally,the resulting solution will be apportioned into vials forlyophilization. Each vial will contain a single dosage (including butnot limited to 10-1000 mg or 100-500 mg) or multiple dosages of thecompound. The lyophilized powder can be stored under appropriateconditions, such as at about 4° C. to room temperature.

Reconstitution of this lyophilized powder with water for injectionprovides a formulation for use in parenteral administration. Forreconstitution, about 1-50 mg, about 5-35 mg, or about 9-30 mg oflyophilized powder, is added per mL of sterile water or other suitablecarrier. The precise amount depends upon the selected compound. Suchamount can be empirically determined.

Topical mixtures are prepared as described for the local and systemicadministration. The resulting mixture may be a solution, suspension,emulsion or the like and are formulated as creams, gels, ointments,emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes,foams, aerosols, irrigations, sprays, suppositories, bandages, dermalpatches or any other formulations suitable for topical administration.

The FTI or pharmaceutical composition having an FTI can be formulated asaerosols for topical application, such as by inhalation (see, e.g., U.S.Pat. Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosolsfor delivery of a steroid useful for treatment of inflammatory diseases,particularly asthma). These formulations for administration to therespiratory tract can be in the form of an aerosol or solution for anebulizer, or as a microfine powder for insufflation, alone or incombination with an inert carrier such as lactose. In such a case, theparticles of the formulation will have diameters of less than 50 micronsor less than 10 microns.

The FTI or pharmaceutical composition having an FTI can be formulatedfor local or topical application, such as for topical application to theskin and mucous membranes, such as in the eye, in the form of gels,creams, and lotions and for application to the eye or for intracisternalor intraspinal application. Topical administration is contemplated fortransdermal delivery and also for administration to the eyes or mucosa,or for inhalation therapies. Nasal solutions of the active compoundalone or in combination with other pharmaceutically acceptableexcipients can also be administered. These solutions, particularly thoseintended for ophthalmic use, may be formulated as 0.01%-10% isotonicsolutions, pH about 5-7, with appropriate salts.

Other routes of administration, such as transdermal patches, and rectaladministration are also contemplated herein. For example, pharmaceuticaldosage forms for rectal administration are rectal suppositories,capsules and tablets for systemic effect. Rectal suppositories are usedherein mean solid bodies for insertion into the rectum which melt orsoften at body temperature releasing one or more pharmacologically ortherapeutically active ingredients. Pharmaceutically acceptablesubstances utilized in rectal suppositories are bases or vehicles andagents to raise the melting point. Examples of bases include cocoabutter (theobroma oil), glycerin gelatin, carbowax (polyoxyethyleneglycol) and appropriate mixtures of mono, di and triglycerides of fattyacids. Combinations of the various bases may be used. Agents to raisethe melting point of suppositories include spermaceti and wax. Rectalsuppositories may be prepared either by the compressed method or bymolding. An exemplary weight of a rectal suppository is about 2 to 3grams. Tablets and capsules for rectal administration are manufacturedusing the same pharmaceutically acceptable substance and by the samemethods as for formulations for oral administration.

The FTI or pharmaceutical composition having an FTI provided herein canbe administered by controlled release means or by delivery devices thatare well known to those of ordinary skill in the art. Examples include,but are not limited to, those described in U.S. Pat. Nos. 3,845,770;3,916,899; 3,536,809; 3,598,123; and 4,008,719, 5,674,533, 5,059,595,5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556, 5,639,480,5,733,566, 5,739,108, 5,891,474, 5,922,356, 5,972,891, 5,980,945,5,993,855, 6,045,830, 6,087,324, 6,113,943, 6,197,350, 6,248,363,6,264,970, 6,267,981, 6,376,461, 6,419,961, 6,589,548, 6,613,358,6,699,500 and 6,740,634, each of which is incorporated herein byreference. Such dosage forms can be used to provide slow orcontrolled-release of FTI using, for example, hydropropylmethylcellulose, other polymer matrices, gels, permeable membranes, osmoticsystems, multilayer coatings, microparticles, liposomes, microspheres,or a combination thereof to provide the desired release profile invarying proportions. Suitable controlled-release formulations known tothose of ordinary skill in the art, including those described herein,can be readily selected for use with the active ingredients providedherein.

All controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledcounterparts. In one embodiment, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. In certain embodiments,advantages of controlled-release formulations include extended activityof the drug, reduced dosage frequency, and increased patient compliance.In addition, controlled-release formulations can be used to affect thetime of onset of action or other characteristics, such as blood levelsof the drug, and can thus affect the occurrence of side (e.g., adverse)effects.

Most controlled-release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release of otheramounts of drug to maintain this level of therapeutic effect over anextended period of time. In order to maintain this constant level ofdrug in the body, the drug must be released from the dosage form at arate that will replace the amount of drug being metabolized and excretedfrom the body. Controlled-release of an active ingredient can bestimulated by various conditions including, but not limited to, pH,temperature, enzymes, water, or other physiological conditions orcompounds.

In certain embodiments, the FTI can be administered using intravenousinfusion, an implantable osmotic pump, a transdermal patch, liposomes,or other modes of administration. In one embodiment, a pump may be used(see, Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald etal., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574(1989). In another embodiment, polymeric materials can be used. In yetanother embodiment, a controlled release system can be placed inproximity of the therapeutic target, i.e., thus requiring only afraction of the systemic dose (see, e.g., Goodson, Medical Applicationsof Controlled Release, vol. 2, pp. 115-138 (1984).

In some embodiments, a controlled release device is introduced into asubject in proximity of the site of inappropriate immune activation or atumor. Other controlled release systems are discussed in the review byLanger (Science 249:1527-1533 (1990). The F can be dispersed in a solidinner matrix, e.g., polymethylmethacrylate, polybutylmethacrylate,plasticized or unplasticized polyvinylchloride, plasticized nylon,plasticized polyethyleneterephthalate, natural rubber, polyisoprene,polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetatecopolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonatecopolymers, hydrophilic polymers such as hydrogels of esters of acrylicand methacrylic acid, collagen, cross-linked polyvinylalcohol andcross-linked partially hydrolyzed polyvinyl acetate, that is surroundedby an outer polymeric membrane, e.g., polyethylene, polypropylene,ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers,ethylene/vinylacetate copolymers, silicone rubbers, polydimethylsiloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride,vinylchloride copolymers with vinyl acetate, vinylidene chloride,ethylene and propylene, ionomer polyethylene terephthalate, butyl rubberepichlorohydrin rubbers, ethylene/vinyl alcohol copolymer,ethylene/vinyl acetate/vinyl alcohol terpolymer, andethylene/vinyloxyethanol copolymer, that is insoluble in body fluids.The active ingredient then diffuses through the outer polymeric membranein a release rate controlling step. The percentage of active ingredientcontained in such parenteral compositions is highly dependent on thespecific nature thereof, as well as the needs of the subject.

The FTI or pharmaceutical composition of FTI can be packaged as articlesof manufacture containing packaging material, a compound orpharmaceutically acceptable salt thereof provided herein, which is usedfor treatment, prevention or amelioration of one or more symptoms orprogression of cancer, including hematological cancers and solid tumors,and a label that indicates that the compound or pharmaceuticallyacceptable salt thereof is used for treatment, prevention oramelioration of one or more symptoms or progression of cancer, includinghematological cancers and solid tumors.

The articles of manufacture provided herein contain packaging materials.Packaging materials for use in packaging pharmaceutical products arewell known to those of skill in the art. See, e.g., U.S. Pat. Nos.5,323,907, 5,052,558 and 5,033,252. Examples of pharmaceutical packagingmaterials include, but are not limited to, blister packs, bottles,tubes, inhalers, pumps, bags, vials, containers, syringes, pens,bottles, and any packaging material suitable for a selected formulationand intended mode of administration and treatment. A wide array offormulations of the compounds and compositions provided herein arecontemplated.

2.3. Dosages

In some embodiments, a therapeutically effective amount of thepharmaceutical composition having an FTI is administered orally orparenterally. In some embodiments, the pharmaceutical composition havingtipifarnib as the active ingredient and is administered orally in anamount of from 1 up to 1500 mg/kg daily, either as a single dose orsubdivided into more than one dose, or more particularly in an amount offrom 10 to 1200 mg/kg daily. In some embodiments, the pharmaceuticalcomposition having tipifarnib as the active ingredient and isadministered orally in an amount of 100 mg/kg daily, 200 mg/kg daily,300 mg/kg daily, 400 mg/kg daily, 500 mg/kg daily, 600 mg/kg daily, 700mg/kg daily, 800 mg/kg daily, 900 mg/kg daily, 1000 mg/kg daily, 1100mg/kg daily, or 1200 mg/kg daily. In some embodiments, the FTI istipifarnib.

In some embodiments, the FTI is administered at a dose of 200-1500 mgdaily. In some embodiments, the FTI is administered at a dose of200-1200 mg daily. In some embodiments, the FTI is administered at adose of 200 mg daily. In some embodiments, the FTI is administered at adose of 300 mg daily. In some embodiments, the FTI is administered at adose of 400 mg daily. In some embodiments, the FTI is administered at adose of 500 mg daily. In some embodiments, the FTI is administered at adose of 600 mg daily. In some embodiments, the FTI is administered at adose of 700 mg daily. In some embodiments, the FTI is administered at adose of 800 mg daily. In some embodiments, the FTI is administered at adose of 900 mg daily. In some embodiments, the FTI is administered at adose of 1000 mg daily. In some embodiments, the FTI is administered at adose of 1100 mg daily. In some embodiments, the FTI is administered at adose of 1200 mg daily. In some embodiments, the FTI is administered at adose of 1300 mg daily. In some embodiments, the FTI is administered at adose of 1400 mg daily. In some embodiments, the FTI is tipifarnib.

In some embodiments, the FTI is administered at a dose of 200-1400 mgb.i.d. (i.e., twice a day). In some embodiments, the FTI is administeredat a dose of 300-1200 mg b.i.d. In some embodiments, the FTI isadministered at a dose of 300-900 mg b.i.d. In some embodiments, the FTIis administered at a dose of 600 mg b.i.d. In some embodiments, the FTIis administered at a dose of 700 mg b.i.d. In some embodiments, the FTIis administered at a dose of 800 mg b.i.d. In some embodiments, the FTIis administered at a dose of 900 mg b.i.d. In some embodiments, the FTIis administered at a dose of 1000 mg b.i.d. In some embodiments, the FTIis administered at a dose of 1100 mg b.i.d. In some embodiments, the FTIis administered at a dose of 1200 mg b.i.d. In some embodiments, the FTIis tipifarnib.

As a person of ordinary skill in the art would understand, the dosagevaries depending on the dosage form employed, condition and sensitivityof the patient, the route of administration, and other factors. Theexact dosage will be determined by the practitioner, in light of factorsrelated to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activeingredient or to maintain the desired effect. Factors which can be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. During a treatmentcycle, the daily dose could be varied. In some embodiments, a startingdosage can be titrated down within a treatment cycle. In someembodiments, a starting dosage can be titrated up within a treatmentcycle. The final dosage can depend on the occurrence of dose limitingtoxicity and other factors. In some embodiments, the FTI is administeredat a starting dose of 300 mg daily and escalated to a maximum dose of400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, or1200 mg daily. In some embodiments, the FTI is administered at astarting dose of 400 mg daily and escalated to a maximum dose of 500 mg,600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, or 1200 mg daily. Insome embodiments, the FTI is administered at a starting dose of 500 mgdaily and escalated to a maximum dose of 600 mg, 700 mg, 800 mg, 900 mg,1000 mg, 1100 mg, or 1200 mg daily. In some embodiments, the FTI isadministered at a starting dose of 600 mg daily and escalated to amaximum dose of 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, or 1200 mgdaily. In some embodiments, the FTI is administered at a starting doseof 700 mg daily and escalated to a maximum dose of 800 mg, 900 mg, 1000mg, 1100 mg, or 1200 mg daily. In some embodiments, the FTI isadministered at a starting dose of 800 mg daily and escalated to amaximum dose of 900 mg, 1000 mg, 1100 mg, or 1200 mg daily. In someembodiments, the FTI is administered at a starting dose of 900 mg dailyand escalated to a maximum dose of 1000 mg, 1100 mg, or 1200 mg daily.The dose escalation can be done at once, or step wise. For example, astarting dose at 600 mg daily can be escalated to a final dose of 1000mg daily by increasing by 100 mg per day over the course of 4 days, orby increasing by 200 mg per day over the course of 2 days, or byincreasing by 400 mg at once. In some embodiments, the FTI istipifarnib.

In some embodiments, the FTI is administered at a relatively highstarting dose and titrated down to a lower dose depending on the patientresponse and other factors. In some embodiments, the FTI is administeredat a starting dose of 1200 mg daily and reduced to a final dose of 1100mg, 1000 mg, 900 mg, 800 mg, 700 mg, 600 mg, 500 mg, 400 mg, or 300 mgdaily. In some embodiments, the FTI is administered at a starting doseof 1100 mg daily and reduced to a final dose of 1000 mg, 900 mg, 800 mg,700 mg, 600 mg, 500 mg, 400 mg, or 300 mg daily. In some embodiments,the FTI is administered at a starting dose of 1000 mg daily and reducedto a final dose of 900 mg, 800 mg, 700 mg, 600 mg, 500 mg, 400 mg, or300 mg daily. In some embodiments, the FTI is administered at a startingdose of 900 mg daily and reduced to a final dose of 800 mg, 700 mg, 600mg, 500 mg, 400 mg, or 300 mg daily. In some embodiments, the FTI isadministered at a starting dose of 800 mg daily and reduced to a finaldose of 700 mg, 600 mg, 500 mg, 400 mg, or 300 mg daily. In someembodiments, the FTI is administered at a starting dose of 600 mg dailyand reduced to a final dose of 500 mg, 400 mg, or 300 mg daily. The dosereduction can be done at once, or step wise. In some embodiments, theFTI is tipifarnib. For example, a starting dose at 900 mg daily can bereduced to a final dose of 600 mg daily by decreasing by 100 mg per dayover the course of 3 days, or by decreasing by 300 mg at once.

A treatment cycle can have different length. In some embodiments, atreatment cycle can be one week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 7 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7months, 8 months, 9 months, 10 months, 11 months, or 12 months. In someembodiments, a treatment cycle is 4 weeks. A treatment cycle can haveintermittent schedule. In some embodiments, a 2-week treatment cycle canhave 5-day dosing followed by 9-day rest. In some embodiments, a 2-weektreatment cycle can have 6-day dosing followed by 8-day rest. In someembodiments, a 2-week treatment cycle can have 7-day dosing followed by7-day rest. In some embodiments, a 2-week treatment cycle can have 8-daydosing followed by 6-day rest. In some embodiments, a 2-week treatmentcycle can have 9-day dosing followed by 5-day rest.

In some embodiments, the FTI is administered daily for 3 of out of 4weeks in repeated 4 week cycles. In some embodiments, the FTI isadministered daily in alternate weeks (one week on, one week off) inrepeated 4 week cycles. In some embodiments, the FTI is administered ata dose of 300 mg b.i.d. orally for 3 of out of 4 weeks in repeated 4week cycles. In some embodiments, the FTI is administered at a dose of600 mg b.i.d. orally for 3 of out of 4 weeks in repeated 4 week cycles.In some embodiments, the FTI is administered at a dose of 900 mg b.i.d.orally in alternate weeks (one week on, one week off) in repeated 4 weekcycles. In some embodiments, the FTI is administered at a dose of 1200mg b.i.d. orally in alternate weeks (days 1-7 and 15-21 of repeated28-day cycles). In some embodiments, the FTI is administered at a doseof 1200 mg b.i.d. orally for days 1-5 and 15-19 out of repeated 28-daycycles.

In some embodiments, a 900 mg b.i.d. tipifarnib alternate week regimencan be used adopted. Under the regimen, patients receive a starting doseof 900 mg, po, b.i.d. on days 1-7 and 15-21 of 28-day treatment cycles.In some embodiments, patients receive two treatment cycles. In someembodiments, patients receive three treatment cycles. In someembodiments, patients receive four treatment cycles. In someembodiments, patients receive five treatment cycles. In someembodiments, patients receive six treatment cycles. In some embodiments,patients receive seven treatment cycles. In some embodiments, patientsreceive eight treatment cycles. In some embodiments, patients receivenine treatment cycles. In some embodiments, patients receive tentreatment cycles. In some embodiments, patients receive eleven treatmentcycles. In some embodiments, patients receive twelve treatment cycles.In some embodiments, patients receive more than twelve treatment cycles.

In the absence of unmanageable toxicities, subjects can continue toreceive the tipifarnib treatment for up to 12 months or longer. The dosecan also be increased to 1200 mg b.i.d. if the subject is tolerating thetreatment well. Stepwise 300 mg dose reductions to controltreatment-related, treatment-emergent toxicities can also be included.

In some other embodiments, tipifarnib is given orally at a dose of 300mg b.i.d. daily for 21 days, followed by 1 week of rest, in 28-daytreatment cycles (21-day schedule; Cheng D T, et al., J Mol Diagn.(2015) 17(3):251-64). In some embodiments, a 5-day dosing ranging from25 to 1300 mg b.i.d. followed by 9-day rest is adopted (5-day schedule;Zujewski J., J Clin Oncol., (2000) February; 18(4):927-41). In someembodiments, a 7-day b.i.d. dosing followed by 7-day rest is adopted(7-day schedule; Lara P N Jr., Anticancer Drugs., (2005) 16(3):317-21;Kirschbaum M H, Leukemia., (2011) October; 25(10):1543-7; Kurzrock, ClinCancer Res (2008), 14(2):509). In the 7-day schedule, the patients canreceive a starting dose of 300 mg b.i.d. with 300 mg dose escalations toa maximum planned dose of 1800 mg b.i.d. In the 7-day schedule study,patients can also receive tipifarnib b.i.d. on days 1-7 and days 15-21of 28-day cycles at doses up to 1600 mg b.i.d.

FTI can inhibit the growth of mammalian tumors when administered as atwice daily dosing schedule. Administration of an FTI in a single dosedaily for one to five days can produce a marked suppression of tumorgrowth lasting out to at least 21 days. In some embodiments, FTI isadministered at a dosage range of 50-400 mg/kg. In some embodiments, FTIis administered at 200 mg/kg. Dosing regimen for specific FTIs are alsowell known in the art (e.g., U.S. Pat. No. 6,838,467, which isincorporated herein by reference in its entirety). For example, suitabledosages for the compounds Arglabin (WO98/28303), perrilyl alcohol (WO99/45712), SCH-66336 (U.S. Pat. No. 5,874,442), L778123 (WO 00/01691),2(S)-[2(S)-[2(R)-amino-3-mercapto]propylamino-3(S)-methyl]-pentyloxy-3-phenylpropionyl-methioninesulfone (WO94/10138), BMS 214662 (WO 97/30992), AZD3409; Pfizercompounds A and B (WO 00/12499 and WO 00/12498) are given in theaforementioned patent specifications which are incorporated herein byreference or are known to or can be readily determined by a personskilled in the art.

In relation to perrilyl alcohol, the medicament may be administered 1-4g per day per 150 lb human patient. Preferably, 1-2 g per day per 150 lbhuman patient. SCH-66336 typically can be administered in a unit dose ofabout 0.1 mg to 100 mg, more preferably from about 1 mg to 300 mgaccording to the particular application. Compounds L778123 and1-(3-chlorophenyl)-4-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-2-piperazinonemay be administered to a human patient in an amount between about 0.1mg/kg of body weight to about 20 mg/kg of body weight per day,preferably between 0.5 mg/kg of bodyweight to about 10 mg/kg of bodyweight per day.

Pfizer compounds A and B may be administered in dosages ranging fromabout 1.0 mg up to about 500 mg per day, preferably from about 1 toabout 100 mg per day in single or divided (i.e. multiple) doses.Therapeutic compounds will ordinarily be administered in daily dosagesranging from about 0.01 to about 10 mg per kg body weight per day, insingle or divided doses. BMS 214662 may be administered in a dosagerange of about 0.05 to 200 mg/kg/day, preferably less than 100 mg/kg/dayin a single dose or in 2 to 4 divided doses.

2.4. Combination Therapies

In some embodiments, the FTI treatment is administered in combinationwith radiotherapy, or radiation therapy. Radiotherapy includes usingγ-rays, X-rays, and/or the directed delivery of radioisotopes to tumorcells. Other forms of DNA damaging factors are also contemplated, suchas microwaves, proton beam irradiation (U.S. Pat. Nos. 5,760,395 and4,870,287; all of which are hereby incorporated by references in theirentireties), and UV-irradiation. It is most likely that all of thesefactors affect a broad range of damage on DNA, on the precursors of DNA,on the replication and repair of DNA, and on the assembly andmaintenance of chromosomes.

In some embodiments, a therapeutically effective amount of thepharmaceutical composition having an FTI is administered thateffectively sensitizes a tumor in a host to irradiation. (U.S. Pat. No.6,545,020, which is hereby incorporated by reference in its entirety).Irradiation can be ionizing radiation and in particular gamma radiation.In some embodiments, the gamma radiation is emitted by linearaccelerators or by radionuclides. The irradiation of the tumor byradionuclides can be external or internal.

Irradiation can also be X-ray radiation. Dosage ranges for X-rays rangefrom daily doses of 50 to 200 roentgens for prolonged periods of time (3to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges forradioisotopes vary widely, and depend on the half-life of the isotope,the strength and type of radiation emitted, and the uptake by theneoplastic cells.

In some embodiments, the administration of the pharmaceuticalcomposition commences up to one month, in particular up to 10 days or aweek, before the irradiation of the tumor. Additionally, irradiation ofthe tumor is fractionated the administration of the pharmaceuticalcomposition is maintained in the interval between the first and the lastirradiation session.

The amount of FTI, the dose of irradiation and the intermittence of theirradiation doses will depend on a series of parameters such as the typeof tumor, its location, the patients' reaction to chemo- or radiotherapyand ultimately is for the physician and radiologists to determine ineach individual case.

In some embodiments, the methods provided herein further includeadministering a therapeutically effective amount of a second activeagent or a support care therapy. The second active agent can be achemotherapeutic agent. A chemotherapeutic agent or drug can becategorized by its mode of activity within a cell, for example, whetherand at what stage they affect the cell cycle. Alternatively, an agentcan be characterized based on its ability to directly cross-link DNA, tointercalate into DNA, or to induce chromosomal and mitotic aberrationsby affecting nucleic acid synthesis.

Examples of chemotherapeutic agents include alkylating agents, such asthiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan,improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines, includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, and uracil mustard;nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine,nimustine, and ranimnustine; antibiotics, such as the enediyneantibiotics (e.g., calicheamicin, especially calicheamicin gammalI andcalicheamicin omegaI1); dynemicin, including dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores, aclacinomysins, actinomycin, authrarnycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolicacid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, and zorubicin; anti-metabolites, such asmethotrexate and 5-fluorouracil (5-FU); folic acid analogues, such asdenopterin, pteropterin, and trimetrexate; purine analogs, such asfludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidineanalogs, such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine;androgens, such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, and testolactone; anti-adrenals, such as mitotane andtrilostane; folic acid replenisher, such as frolinic acid; aceglatone;aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSKpolysaccharidecomplex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g.,paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine;platinum coordination complexes, such as cisplatin, oxaliplatin, andcarboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;mitoxantrone; vincristine; vinorelbine; novantrone; teniposide;edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan(e.g., CPT-11); topoisomerase inhibitor RFS 2000;difluorometlhylornithine (DMFO); retinoids, such as retinoic acid;capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine,navelbine, transplatinum, and pharmaceutically acceptable salts, acids,or derivatives of any of the above.

The second active agents can be large molecules (e.g., proteins) orsmall molecules (e.g., synthetic inorganic, organometallic, or organicmolecules). In some embodiments, the second active agent is aDNA-hypomethylating agent, a therapeutic antibody that specificallybinds to a cancer antigen, a hematopoietic growth factor, cytokine,anti-cancer agent, antibiotic, cox-2 inhibitor, immunomodulatory agent,anti-thymocyte globulin, immunosuppressive agent, corticosteroid or apharmacologically active mutant or derivative thereof.

In some embodiments, the second active agent is a DNA hypomethylatingagent, such as a cytidine analog (e.g., azacitidine) or a5-azadeoxycytidine (e.g. decitabine). In some embodiments, the secondactive agent is a cytoreductive agent, including but not limited toInduction, Topotecan, Hydrea, PO Etoposide, Lenalidomide, LDAC, andThioguanine. In some embodiments, the second active agent isMitoxantrone, Etoposide, Cytarabine, or Valspodar. In some embodiment,the second active agent is Mitoxantrone plus Valspodar, Etoposide plusValspodar, or Cytarabine plus Valspodar. In some embodiment, the secondactive agent is idarubicin, fludarabine, topotecan, or ara-C. In someother embodiments, the second active agent is idarubicin plus ara-C,fludarabine plus ara-C, mitoxantrone plus ara-C, or topotecan plusara-C. In some embodiments, the second active agent is a quinine. Othercombinations of the agents specified above can be used, and the dosagescan be determined by the physician.

In some embodiments, the second active agent is an immunotherapy agent.In some embodiments, the second active agent is anti-PD1 antibody oranti-PDL1 antibody.

In some embodiments, it is contemplated that the second active agent orsecond therapy used in combination with an FTI can be administeredbefore, at the same time, or after the FTI treatment. In someembodiments, the second active agent or second therapy used incombination with an FTI can be administered before the FTI treatment. Insome embodiments, the second active agent or second therapy used incombination with an FTI can be administered at the same time as FTItreatment. In some embodiments, the second active agent or secondtherapy used in combination with an FTI can be administered after theFTI treatment.

The FTI treatment can also be administered in combination with a bonemarrow transplant. In some embodiments, the FTI is administered beforethe bone marrow transplant. In other embodiments, the FTI isadministered after the bone marrow transplant.

3. Biomarkers for FTI Treatment

Provided herein are methods of selection of squamous cell carcinoma ofthe head and neck (SCCHN) and lung squamous cell carcinoma (lung SCC)patients for treatment with a farnesyltransferase inhibitor (FTI).Farnesyltransferase (FTase) have crucial roles in the post-translationalmodifications of Ras proteins. FTIs are a class of biologically activeanticancer drugs that inhibit farnesylation of a wide range of targetproteins, including Ras. The Ras proteins play a pivotal role in thetransduction of cell growth-stimulating signals, and mutation of the rasgene leads to constant activation of the protein, ultimately resultingin uncontrolled cell proliferation. The high prevalence of mutated rasgenes, found in 30% of all human cancers, makes this pathway anattractive target for anticancer drug development. A way of interferingwith Ras function is the inhibition of FTase, the enzyme coupling a15-carbon isoprenyl group to Ras proteins, by FTIs. The FTIs block Rasactivation through inhibition of FTase, ultimately resulting in cellgrowth arrest. Thus, it was predicted that FTIs would be effectivetherapeutic agents in the treatment of cancer.

However, no correlation between ras mutations and response to FTIs wasdemonstrated in past clinical studies (Karp et al. Blood 97:3361-3369(2001); and US. Patent Pub. 20070048782)). While several early clinicalstudies focused on cancers that exhibited high frequencies of rasmutations, the response rate was disappointingly low in those trials.(Mesa Lancet Oncol 6:279-286 (2006); Rao et al. J Clin Oncol22:3950-3957 (2004))

Early studies of tipifarnib, an FTI, were conducted in poor risk andpreviously untreated AML patients (CTEP-20 phase II), and AML patientswith relapsed/refractory AML (INT-17 Phase II). A phase III study oftipifarnib versus best supportive care (BSC) failed to demonstrateimprovement in overall survival. Multiple gene/proteins have beenassociated in the literature with the activity of FTI (AKAP13, mDIA,etc.) (Raponi et al. Clin Cancer Res. 13:2254-60 (2007); Kamasani et al.Cancer Biology & Therapy, 6:1418-1423 (2007)), and analyses of geneexpression profiling in bone marrow samples from 2 AML studies (CTEP-20,INT-17) identified the ratio of the expression of 2 genes: RASGRP1 (Tcell signal transducer) and APTX (DNA repair protein) as a potentialbiomarker of tipifarnib's activity in AML (Raponi et al. Blood.111:2589-96 (2008)). However, a subsequent prospective study using the2-gene ratio in bone marrow blasts as inclusion criterion failed todemonstrate significant clinical benefit of tipifarnib in AML (Lancet etal. Blood (ASH) 120: Abstract 1508 (2012)).

The present invention identifies HRAS mutations as biomarkers associatedwith better prognosis for an FTI treatment, and novel methods areprovided herein for patient selection for an FTI treatment. The HRASmutations identified in the instant application are specificallyassociated with the clinical benefit of an FTI treatment, but not withthe clinical benefit of agents of other standard chemotherapies.

As disclosed herein, the methods can also be used in connection withother patient stratification approaches to further increase the responserate of a patient population to an FTI treatment. For example, in someembodiments, the methods provided herein further include determining themutation status of the HRAS gene and selecting a patient for an FTItreatment, as described in greater detail below. In some embodiments,the methods provided herein further include determining the mutationstatus of the ras genes and selecting a patient for an FTI treatmentwhen the patient has an HRAS mutation with wild type K-ras and wild typeN-ras. In other embodiments, the methods provided herein can furtherinclude using the 2 gene ratio between RASGRP1 and APTX as additionalpatient selection criterion for an FTI treatment (U.S. Pat. No.7,932,036, which is hereby incorporated by reference in its entirety).Methods described herein or otherwise known in the art can be used todetermine the mutation status of the ras gene, such as the HRAS gene. Insome embodiments, the mutation status of a ras gene, such as HRAS, canbe determined by NGS.

In some embodiments, the methods provided herein include determining theexpression level of a biomarker. In some embodiments, the expressionlevel of a biomarker can be the protein level of the biomarker. In someembodiments, the expression level of a biomarker can be the RNA level ofthe biomarker. Any method as described herein or otherwise known in theart to determine the protein level or RNA level of a gene can be usedfor determining the expression level of a biomarker in presentinvention.

Exemplary methods of detecting or quantitating mRNA levels include butare not limited to PCR-based methods, northern blots, ribonucleaseprotection assays, and the like. The mRNA sequence (e.g., the mRNA of abiomarker, such as CRBN or a CAP, or a fragment thereof) can be used toprepare a probe that is at least partially complementary. The probe canthen be used to detect the mRNA sequence in a sample, using any suitableassay, such as PCR-based methods, Northern blotting, a dipstick assay,and the like.

The commonly used methods known in the art for the quantification ofmRNA expression in a sample include northern blotting and in situhybridization (Parker &Barnes, Methods in Molecular Biology 106:247-283(1999)); RNAse protection assays (Hod, Biotechniques 13:852-854 (1992));and polymerase chain reaction (PCR) (Weis et ah, Trends in Genetics8:263-264 (1992)). Alternatively, antibodies may be employed that canrecognize specific duplexes, including DNA duplexes, RNA duplexes, andDNA-RNA hybrid duplexes or DNA-protein duplexes. Representative methodsfor sequencing-based gene expression analysis include Serial Analysis ofGene Expression (SAGE), and gene expression analysis by massivelyparallel signature sequencing (MPSS).

A sensitive and flexible quantitative method is PCR. Examples of PCRmethods can be found in the literature. Examples of PCR assays can befound in U.S. Pat. No. 6,927,024, which is incorporated by referenceherein in its entirety. Examples of RT-PCR methods can be found in U.S.Pat. No. 7,122,799, which is incorporated by reference herein in itsentirety. A method of fluorescent in situ PCR is described in U.S. Pat.No. 7,186,507, which is incorporated by reference herein in itsentirety.

It is noted, however, that other nucleic acid amplification protocols(i.e., other than PCR) may also be used in the nucleic acid analyticalmethods described herein. For example, suitable amplification methodsinclude ligase chain reaction (see, e.g., Wu & Wallace, Genomics4:560-569, 1988); strand displacement assay (see, e.g., Walker et al.,Proc. Natl. Acad. Sci. USA 89:392-396, 1992; U.S. Pat. No. 5,455,166);and several transcription-based amplification systems, including themethods described in U.S. Pat. Nos. 5,437,990; 5,409,818; and 5,399,491;the transcription amplification system (TAS) (Kwoh et al., Proc. Natl.Acad. Sci. USA 86: 1173-1177, 1989); and self-sustained sequencereplication (3SR) (Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874-1878, 1990; WO 92/08800). Alternatively, methods that amplify theprobe to detectable levels can be used, such as Q-replicaseamplification (Kramer & Lizardi, Nature 339:401-402, 1989; Lomeli etal., Clin. Chem. 35: 1826-1831, 1989). A review of known amplificationmethods is provided, for example, by Abramson and Myers in CurrentOpinion in Biotechnology 4:41-47 (1993).

mRNA may be isolated from the starting tissue sample. General methodsfor mRNA extraction are well known in the art and are disclosed instandard textbooks of molecular biology, including Ausubel et al.,Current Protocols of Molecular Biology, John Wiley and Sons (1997). Inparticular, RNA isolation can be performed using purification kit,buffer set and protease from commercial manufacturers, such as Qiagen,according to the manufacturer's instructions. For example, total RNAfrom cells in culture can be isolated using Qiagen RNeasy mini-columns.Other commercially available RNA isolation kits include MASTERPURE®Complete DNA and RNA Purification Kit (EPICENTRE®, Madison, Wis.), andParaffin Block RNA Isolation Kit (Ambion, Inc.). Total RNA from tissuesamples can be isolated using RNA Stat-60 (Tel-Test). RNA prepared fromtumor can be isolated, for example, by cesium chloride density gradientcentrifugation.

In some embodiments, the first step in gene expression profiling by PCRis the reverse transcription of the RNA template into cDNA, followed byits exponential amplification in a PCR reaction. In other embodiments, acombined reverse-transcription-polymerase chain reaction (RT-PCR)reaction may be used, e.g., as described in U.S. Pat. Nos. 5,310,652;5,322,770; 5,561,058; 5,641,864; and 5,693,517. The two commonly usedreverse transcriptases are avilo myeloblastosis virus reversetranscriptase (AMV-RT) and Moloney murine leukemia virus reversetranscriptase (MMLV-RT). The reverse transcription step is typicallyprimed using specific primers, random hexamers, or oligo-dT primers,depending on the circumstances and the goal of expression profiling. Forexample, extracted RNA can be reverse-transcribed using a GENEAMP™ RNAPCR kit (Perkin Elmer, Calif., USA), following the manufacturer'sinstructions. The derived cDNA can then be used as a template in thesubsequent PCR reaction.

In some embodiments, Real-Time Reverse Transcription-PCR (qRT-PCR) canbe used for both the detection and quantification of RNA targets(Bustin, et al., 2005, Clin. Sci., 109:365-379). Examples ofqRT-PCR-based methods can be found, for example, in U.S. Pat. No.7,101,663, which is incorporated by reference herein in its entirety.Instruments for real-time PCR, such as the Applied Biosystems 7500, areavailable commercially, as are the reagents, such as TaqMan SequenceDetection chemistry.

For example, TagManx Gene Expression Assays can be used, following themanufacturer's instructions. These kits are pre-formulated geneexpression assays for rapid, reliable detection and quantification ofhuman, mouse and rat mRNA transcripts. TaqMan® or 5′-nuclease assay, asdescribed in U.S. Pat. Nos. 5,210,015; 5,487,972; and 5,804,375; andHolland et al., 1988, Proc. Natl. Acad. Sci. USA 88:7276-7280, can beused. TAQMAN® PCR typically utilizes the 5′-nuclease activity of Taq orTth polymerase to hydrolyze a hybridization probe bound to its targetamplicon, but any enzyme with equivalent 5′ nuclease activity can beused. Two oligonucleotide primers are used to generate an amplicontypical of a PCR reaction. A third oligonucleotide, or probe, isdesigned to detect nucleotide sequence located between the two PCRprimers. The probe is non-extendible by Taq DNA polymerase enzyme, andis labeled with a reporter fluorescent dye and a quencher fluorescentdye. Any laser-induced emission from the reporter dye is quenched by thequenching dye when the two dyes are located close together as they areon the probe. During the amplification reaction, the Taq DNA polymeraseenzyme cleaves the probe in a template-dependent manner. The resultantprobe fragments disassociate in solution, and signal from the releasedreporter dye is free from the quenching effect of the secondfluorophore. One molecule of reporter dye is liberated for each newmolecule synthesized, and detection of the unquenched reporter dyeprovides the basis for quantitative interpretation of the data.

Any method suitable for detecting degradation product can be used in a5′ nuclease assay. Often, the detection probe is labeled with twofluorescent dyes, one of which is capable of quenching the fluorescenceof the other dye. The dyes are attached to the probe, preferably oneattached to the 5′ terminus and the other is attached to an internalsite, such that quenching occurs when the probe is in an unhybridizedstate and such that cleavage of the probe by the 5′ to 3′ exonucleaseactivity of the DNA polymerase occurs in between the two dyes.

Amplification results in cleavage of the probe between the dyes with aconcomitant elimination of quenching and an increase in the fluorescenceobservable from the initially quenched dye. The accumulation ofdegradation product is monitored by measuring the increase in reactionfluorescence. U.S. Pat. Nos. 5,491,063 and 5,571,673, both incorporatedherein by reference, describe alternative methods for detecting thedegradation of probe which occurs concomitant with amplification.5′-Nuclease assay data may be initially expressed as Ct, or thethreshold cycle. As discussed above, fluorescence values are recordedduring every cycle and represent the amount of product amplified to thatpoint in the amplification reaction. The point when the fluorescentsignal is first recorded as statistically significant is the thresholdcycle (Ct).

To minimize errors and the effect of sample-to-sample variation, PCR isusually performed using an internal standard. The ideal internalstandard is expressed at a constant level among different tissues, andis unaffected by the experimental treatment. RNAs most frequently usedto normalize patterns of gene expression are mRNAs for the housekeepinggenes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and P-actin.

PCR primers and probes are designed based upon intron sequences presentin the gene to be amplified. In this embodiment, the first step in theprimer/probe design is the delineation of intron sequences within thegenes. This can be done by publicly available software, such as the DNABLAT software developed by Kent, W., Genome Res. 12(4):656-64 (2002), orby the BLAST software including its variations. Subsequent steps followwell established methods of PCR primer and probe design.

In order to avoid non-specific signals, it can be important to maskrepetitive sequences within the introns when designing the primers andprobes. This can be easily accomplished by using the Repeat Maskerprogram available on-line through the Baylor College of Medicine, whichscreens DNA sequences against a library of repetitive elements andreturns a query sequence in which the repetitive elements are masked.The masked intron sequences can then be used to design primer and probesequences using any commercially or otherwise publicly availableprimer/probe design packages, such as Primer Express (AppliedBiosystems); MGB assay-by-design (Applied Biosystems); Primer3 (Rozenand Skaletsky (2000) Primer3 on the WWW for general users and forbiologist programmers. In: Krawetz S, Misener S (eds) BioinformaticsMethods and Protocols: Methods in Molecular Biology. Humana Press,Totowa, N.J., pp 365-386).

Factors considered in PCR primer design include primer length, meltingtemperature (Tm), and G/C content, specificity, complementary primersequences, and 3′-end sequence. In general, optimal PCR primers aregenerally 17-30 bases in length, and contain about 20-80%, such as, forexample, about 50-60% G+C bases. Tm's between 50 and 80° C., e.g. about50 to 70° C. are typically preferred. For further guidelines for PCRprimer and probe design see, e.g. Dieffenbach et ah, “General Conceptsfor PCR Primer Design” in: PCR Primer, A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York, 1995, pp. 133-155; Innis and Gelfand,“Optimization of PCRs” in: PCR Protocols, A Guide to Methods andApplications, CRC Press, London, 1994, pp. 5-11; and Plasterer, T. N.Primerselect: Primer and probe design. Methods Mol. Biol. 70:520-527(1997), the entire disclosures of which are hereby expresslyincorporated by reference.

An exemplary PCR program, for example, is 50° C. for 2 minutes, 95° C.for 10 minutes, 40 cycles of 95° C. for 15 seconds, then 60° C. for 1minute. To determine the cycle number at which the fluorescence signalassociated with a particular amplicon accumulation crosses the threshold(referred to as the CT), the data can be analyzed, for example, using a7500 Real-Time PCR System Sequence Detection software v1.3 using thecomparative CT relative quantification calculation method. Using thismethod, the output is expressed as a fold-change of expression levels.In some embodiments, the threshold level can be selected to beautomatically determined by the software. In some embodiments, thethreshold level is set to be above the baseline but sufficiently low tobe within the exponential growth region of an amplification curve.

RNA-Seq, also called Whole Transcriptome Shotgun Sequencing (WTSS)refers to the use of high-throughput sequencing technologies to sequencecDNA in order to get information about a sample's RNA content.Publications describing RNA-Seq include: Wang et al., Nature ReviewsGenetics 10 (1): 57-63 (January 2009); Ryan et al. BioTechniques 45 (1):81-94 (2008); and Maher et al., Nature 458 (7234): 97-101 (January2009); which are hereby incorporated in their entirety.

Differential gene expression can also be identified, or confirmed usingthe microarray technique. In this method, polynucleotide sequences ofinterest (including cDNAs and oligonucleotides) are plated, or arrayed,on a microchip substrate. The arrayed sequences are then hybridized withspecific DNA probes from cells or tissues of interest.

In an embodiment of the microarray technique, PCR amplified inserts ofcDNA clones are applied to a substrate in a dense array. Preferably atleast 10,000 nucleotide sequences are applied to the substrate. Themicroarrayed genes, immobilized on the microchip at 10,000 elementseach, are suitable for hybridization under stringent conditions.Fluorescently labeled cDNA probes may be generated through incorporationof fluorescent nucleotides by reverse transcription of RNA extractedfrom tissues of interest. Labeled cDNA probes applied to the chiphybridize with specificity to each spot of DNA on the array. Afterstringent washing to remove non-specifically bound probes, the chip isscanned by confocal laser microscopy or by another detection method,such as a CCD camera. Quantitation of hybridization of each arrayedelement allows for assessment of corresponding mRNA abundance. With dualcolor fluorescence, separately labeled cDNA probes generated from twosources of RNA are hybridized pairwise to the array. The relativeabundance of the transcripts from the two sources corresponding to eachspecified gene is thus determined simultaneously. The miniaturized scaleof the hybridization affords a convenient and rapid evaluation of theexpression pattern for large numbers of genes. Such methods have beenshown to have the sensitivity required to detect rare transcripts, whichare expressed at a few copies per cell, and to reproducibly detect atleast approximately two-fold differences in the expression levels(Schena et al., Proc. Natl. Acad. Sci. USA 93(2): 106-149 (1996)).Microarray analysis can be performed by commercially availableequipment, following manufacturer's protocols, such as by using theAffymetrix GENCHIP™ technology, or Incyte's microarray technology.

Serial analysis of gene expression (SAGE) is a method that allows thesimultaneous and quantitative analysis of a large number of genetranscripts, without the need of providing an individual hybridizationprobe for each transcript. First, a short sequence tag (about 10-14 bp)is generated that contains sufficient information to uniquely identify atranscript, provided that the tag is obtained from a unique positionwithin each transcript. Then, many transcripts are linked together toform long serial molecules, that can be sequenced, revealing theidentity of the multiple tags simultaneously. The expression pattern ofany population of transcripts can be quantitatively evaluated bydetermining the abundance of individual tags, and identifying the genecorresponding to each tag. For more details see, e.g. Velculescu et ah,Science 270:484-487 (1995); and Velculescu et al, Cell 88:243-51 (1997).

The MassARRAY (Sequenom, San Diego, Calif.) technology is an automated,high-throughput method of gene expression analysis using massspectrometry (MS) for detection. According to this method, following theisolation of RNA, reverse transcription and PCR amplification, the cDNAsare subjected to primer extension. The cDNA-derived primer extensionproducts are purified, and dispensed on a chip array that is pre-loadedwith the components needed for MALTI-TOF MS sample preparation. Thevarious cDNAs present in the reaction are quantitated by analyzing thepeak areas in the mass spectrum obtained.

mRNA level can also be measured by an assay based on hybridization. Atypical mRNA assay method can contain the steps of 1) obtainingsurface-bound subject probes; 2) hybridization of a population of mRNAsto the surface-bound probes under conditions sufficient to provide forspecific binding (3) post-hybridization washes to remove nucleic acidsnot bound in the hybridization; and (4) detection of the hybridizedmRNAs. The reagents used in each of these steps and their conditions foruse may vary depending on the particular application.

Any suitable assay platform can be used to determine the mRNA level in asample. For example, an assay can be in the form of a dipstick, amembrane, a chip, a disk, a test strip, a filter, a microsphere, aslide, a multiwell plate, or an optical fiber. An assay system can havea solid support on which a nucleic acid corresponding to the mRNA isattached. The solid support can have, for example, a plastic, silicon, ametal, a resin, glass, a membrane, a particle, a precipitate, a gel, apolymer, a sheet, a sphere, a polysaccharide, a capillary, a film aplate, or a slide. The assay components can be prepared and packagedtogether as a kit for detecting an mRNA.

The nucleic acid can be labeled, if desired, to make a population oflabeled mRNAs. In general, a sample can be labeled using methods thatare well known in the art (e.g., using DNA ligase, terminal transferase,or by labeling the RNA backbone, etc.; see, e.g., Ausubel, et al., ShortProtocols in Molecular Biology, 3rd ed., Wiley & Sons 1995 and Sambrooket al., Molecular Cloning: A Laboratory Manual, Third Edition, 2001 ColdSpring Harbor, N.Y.). In some embodiments, the sample is labeled withfluorescent label. Exemplary fluorescent dyes include but are notlimited to xanthene dyes, fluorescein dyes, rhodamine dyes, fluoresceinisothiocyanate (FITC), 6 carboxyfluorescein (FAM), 6carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 6 carboxy 4′, 5′dichloro 2′, 7′ dimethoxyfluorescein (JOE or J), N,N,N′,N′ tetramethyl 6carboxyrhodamine (TAMRA or T), 6 carboxy X rhodamine (ROX or R), 5carboxyrhodamine 6G (R6G5 or G5), 6 carboxyrhodamine 6G (R6G6 or G6),and rhodamine 110; cyanine dyes, e.g. Cy3, Cy5 and Cy7 dyes; Alexa dyes,e.g. Alexa-fluor-555; coumarin, Diethylaminocoumarin, umbelliferone;benzimide dyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red;ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes;porphyrin dyes; polymethine dyes, BODIPY dyes, quinoline dyes, Pyrene,Fluorescein Chlorotriazinyl, R110, Eosin, JOE, R6G,Tetramethylrhodamine, Lissamine, ROX, Napthofluorescein, and the like.

Hybridization can be carried out under suitable hybridizationconditions, which may vary in stringency as desired. Typical conditionsare sufficient to produce probe/target complexes on a solid surfacebetween complementary binding members, i.e., between surface-boundsubject probes and complementary mRNAs in a sample. In certainembodiments, stringent hybridization conditions can be employed.

Hybridization is typically performed under stringent hybridizationconditions. Standard hybridization techniques (e.g. under conditionssufficient to provide for specific binding of target mRNAs in the sampleto the probes) are described in Kallioniemi et al., Science 258:818-821(1992) and WO 93/18186. Several guides to general techniques areavailable, e.g., Tijssen, Hybridization with Nucleic Acid Probes, PartsI and II (Elsevier, Amsterdam 1993). For descriptions of techniquessuitable for in situ hybridizations, see Gall et al. Meth. Enzymol.,21:470-480 (1981); and Angerer et al. in Genetic Engineering: Principlesand Methods (Setlow and Hollaender, Eds.) Vol 7, pgs 43-65 (PlenumPress, New York 1985). Selection of appropriate conditions, includingtemperature, salt concentration, polynucleotide concentration,hybridization time, stringency of washing conditions, and the like willdepend on experimental design, including source of sample, identity ofcapture agents, degree of complementarity expected, etc., and may bedetermined as a matter of routine experimentation for those of ordinaryskill in the art. Those of ordinary skill will readily recognize thatalternative but comparable hybridization and wash conditions can beutilized to provide conditions of similar stringency.

After the mRNA hybridization procedure, the surface boundpolynucleotides are typically washed to remove unbound nucleic acids.Washing may be performed using any convenient washing protocol, wherethe washing conditions are typically stringent, as described above. Thehybridization of the target mRNAs to the probes is then detected usingstandard techniques.

IHC staining of tissue sections has been shown to be a reliable methodof assessing or detecting presence of proteins in a sample.Immunohistochemistry techniques utilize an antibody to probe andvisualize cellular antigens in situ, generally by chromogenic orfluorescent methods. Thus, antibodies or antisera, preferably polyclonalantisera, and most preferably monoclonal antibodies specific for eachmarker are used to detect expression. As discussed in greater detailbelow, the antibodies can be detected by direct labeling of theantibodies themselves, for example, with radioactive labels, fluorescentlabels, hapten labels such as, biotin, or an enzyme such as horse radishperoxidase or alkaline phosphatase. Alternatively, unlabeled primaryantibody is used in conjunction with a labeled secondary antibody,comprising antisera, polyclonal antisera or a monoclonal antibodyspecific for the primary antibody.

Immunohistochemistry protocols and kits are well known in the art andare commercially available. Automated systems for slide preparation andIHC processing are available commercially. The Ventana® BenchMark XTsystem is an example of such an automated system.

Standard immunological and immunoassay procedures can be found in Basicand Clinical Immunology (Stites & Terr eds., 7th ed. 1991). Moreover,the immunoassays can be performed in any of several configurations,which are reviewed extensively in Enzyme Immunoassay (Maggio, ed.,1980); and Harlow & Lane, supra. For a review of the generalimmunoassays, see also Methods in Cell Biology: Antibodies in CellBiology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology(Stites & Ten, eds., 7th ed. 1991).

Commonly used assays to detect protein level of a biomarker includenoncompetitive assays, e.g., sandwich assays, and competitive assays.Typically, an assay such as an ELISA assay can be used. ELISA assays areknown in the art, e.g., for assaying a wide variety of tissues andsamples, including blood, plasma, serum or bone marrow.

A wide range of immunoassay techniques using such an assay format areavailable, see, e.g., U.S. Pat. Nos. 4,016,043, 4,424,279, and4,018,653, which are hereby incorporated by reference in theirentireties. These include both single-site and two-site or “sandwich”assays of the non-competitive types, as well as in the traditionalcompetitive binding assays. These assays also include direct binding ofa labeled antibody to a target biomarker. Sandwich assays are commonlyused assays. A number of variations of the sandwich assay techniqueexist. For example, in a typical forward assay, an unlabelled antibodyis immobilized on a solid substrate, and the sample to be tested broughtinto contact with the bound molecule. After a suitable period ofincubation, for a period of time sufficient to allow formation of anantibody-antigen complex, a second antibody specific to the antigen,labeled with a reporter molecule capable of producing a detectablesignal is then added and incubated, allowing time sufficient for theformation of another complex of antibody-antigen-labeled antibody. Anyunreacted material is washed away, and the presence of the antigen isdetermined by observation of a signal produced by the reporter molecule.The results may either be qualitative, by simple observation of thevisible signal, or may be quantitated by comparing with a control samplecontaining known amounts of biomarker.

Variations on the forward assay include a simultaneous assay, in whichboth sample and labeled antibody are added simultaneously to the boundantibody. These techniques are well known to those skilled in the art,including any minor variations as will be readily apparent. In a typicalforward sandwich assay, a first antibody having specificity for thebiomarker is either covalently or passively bound to a solid surface.The solid surface may be glass or a polymer, the most commonly usedpolymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinylchloride, or polypropylene. The solid supports may be in the form oftubes, beads, discs of microplates, or any other surface suitable forconducting an immunoassay. The binding processes are well-known in theart and generally consist of cross-linking covalently binding orphysically adsorbing, the polymer-antibody complex is washed inpreparation for the test sample. An aliquot of the sample to be testedis then added to the solid phase complex and incubated for a period oftime sufficient (e.g. 2-40 minutes or overnight if more convenient) andunder suitable conditions (e.g., from room temperature to 40° C. such asbetween 25° C. and 32° C. inclusive) to allow binding of any subunitpresent in the antibody. Following the incubation period, the antibodysubunit solid phase is washed and dried and incubated with a secondantibody specific for a portion of the biomarker. The second antibody islinked to a reporter molecule which is used to indicate the binding ofthe second antibody to the molecular marker.

In some embodiments, flow cytometry (FACS) can be used to detect theprotein level of a biomarker. Surface proteins can be detected usingantibodies against specific biomarkers. The flow cytometer detects andreports the intensity of the fluorichrome-tagged antibody, whichindicates the expression level of the biomarker. Non-fluorescentcytoplasmic proteins can also be observed by staining permeablizedcells. The stain can either be a fluorescence compound able to bind tocertain molecules, or a fluorichrome-tagged antibody to bind themolecule of choice.

An alternative method involves immobilizing the target biomarkers in thesample and then exposing the immobilized target to specific antibody,which may or may not be labeled with a reporter molecule. Depending onthe amount of target and the strength of the reporter molecule signal, abound target may be detectable by direct labeling with the antibody.Alternatively, a second labeled antibody, specific to the first antibodyis exposed to the target-first antibody complex to form a target-firstantibody-second antibody tertiary complex. The complex is detected bythe signal emitted by a labeled reporter molecule.

In the case of an enzyme immunoassay, an enzyme is conjugated to thesecond antibody, generally by means of glutaraldehyde or periodate. Aswill be readily recognized, however, a wide variety of differentconjugation techniques exist, which are readily available to the skilledartisan. Commonly used enzymes include horseradish peroxidase, glucoseoxidase, beta-galactosidase, and alkaline phosphatase, and other arediscussed herein. The substrates to be used with the specific enzymesare generally chosen for the production, upon hydrolysis by thecorresponding enzyme, of a detectable color change. Examples of suitableenzymes include alkaline phosphatase and peroxidase. It is also possibleto employ fluorogenic substrates, which yield a fluorescent productrather than the chromogenic substrates noted above. In all cases, theenzyme-labeled antibody is added to the first antibody-molecular markercomplex, allowed to bind, and then the excess reagent is washed away. Asolution containing the appropriate substrate is then added to thecomplex of antibody-antigen-antibody. The substrate will react with theenzyme linked to the second antibody, giving a qualitative visualsignal, which may be further quantitated, usuallyspectrophotometrically, to give an indication of the amount of biomarkerwhich was present in the sample. Alternately, fluorescent compounds,such as fluorescein and rhodamine, can be chemically coupled toantibodies without altering their binding capacity. When activated byillumination with light of a particular wavelength, thefluorochrome-labeled antibody adsorbs the light energy, inducing a stateto excitability in the molecule, followed by emission of the light at acharacteristic color visually detectable with a light microscope. As inthe EIA, the fluorescent labeled antibody is allowed to bind to thefirst antibody-molecular marker complex. After washing off the unboundreagent, the remaining tertiary complex is then exposed to the light ofthe appropriate wavelength, the fluorescence observed indicates thepresence of the molecular marker of interest. Immunofluorescence and EIAtechniques are both very well established in the art and are discussedherein.

In some embodiments, the methods provided herein include determining theprotein levels of two or more of these biomarkers. In some embodiments,the methods include determining the protein levels of three or more ofthese biomarkers. In some embodiments, the methods include determiningthe protein levels of four or more of these biomarkers. In someembodiments, the methods include determining the protein levels of fiveof these biomarkers.

3.4. Reference Levels and Reference Ratios

In some embodiments, the reference expression level of a biomarker orthe reference ratio between expression levels of two biomarkers can bedetermined based on statistical analysis of data from previous clinicaltrials, including outcome of a group of patients, namely, the patients'responsiveness to an FTI treatment, as well as the expression levels ofthe biomarker or ratio of expression levels between biomarkers of thegroup of patients. A number of statistical methods are well known in theart to determine the reference level (or referred to as the “cut-offvalue”) of one or more biomarkers when used to predict theresponsiveness of a patient to a particular treatment, or to stratifypatients for a particular treatment.

One method of the invention includes analyzing gene expression profilesfor biomarkers identified herein that distinguish responder fromnon-responder to determine the reference expression level for one ormore biomarkers. Comparisons between responders and non-responders canbe performed using the Mann-Whitney U-test, Chi-square test, or Fisher'sExact test. Analysis of descriptive statistics and comparisons can beperformed using SigmaStat Software (Systat Software, Inc., San Jose,Calif., USA).

In some embodiments, a classification and regression tree (CART)analysis can be adopted to determine the reference level. CART analysisis based on a binary recursive partitioning algorithm and allows for thediscovery of complex predictor variable interactions that may not beapparent with more traditional methods, such as multiple linearregression. Binary recursive partitioning refers to the analysis thatis: 1) binary, meaning there were two possible outcome variables, namely“responders” and “non-responders,” with the effect of splitting patientsinto 2 groups; 2) recursive, meaning the analysis can be performedmultiple times; and 3) partitioned, meaning the entire data set can besplit into sections. This analysis also has the ability to eliminatepredictor variables with poor performance. The classification tree canbe built using Salford Predictive Modeler v6.6 (Salford Systems, SanDiego, Calif., USA).

Articles of this invention are representations of the gene expressionprofiles useful for predicting the responsiveness of a cancer patient toan FTI treatment that are reduced to a medium that can be automaticallyread such as computer readable media (magnetic, optical, and the like).The articles can also include instructions for assessing the geneexpression profiles in such media. For example, the articles maycomprise a CD-ROM having computer instructions for comparing geneexpression profiles of biomarkers described above. The articles may alsohave gene expression profiles digitally recorded therein so that theymay be compared with gene expression data from patient samples.Alternatively, the profiles can be recorded in differentrepresentational format. Clustering algorithms such as thoseincorporated in “OMNIVIZ” and “TREE VIEW” computer programs mentionedabove can best assist in the visualization of such data.

Receiver Operator Characteristic (ROC) analysis can be utilized todetermine the reference expression level, or reference expression ratio,or test the overall predictive value of individual genes and/ormultigene classifiers. A review of the ROC analysis can be found inSoreide, J Clin Pathol 10.1136 (2008), which is herby incorporated byreference in its entirety.

The reference level can be determined from the ROC curve of the trainingset to ensure both high sensitivity and high specificity. To determinehow many biomarkers are needed to be included in the predictor,leave-one-out cross validation (LOOCV) can be used. The response scoresfor the ‘left-out’ samples based on different numbers of genes arerecorded. The performances of the predictors with different numbers ofgenes can be assessed based on misclassification error rate,sensitivity, specificity, p values measuring the separation ofKaplan-Meier curves of the two predicted groups.

The Top Scoring Pair (TSP) algorithm first introduced by Geman et al.(2004) can be used. In essence, the algorithm ranks all the gene pairs(genes i and j) based on the absolute difference (Dij) in the frequencyof event where gene i has higher expression value than gene j in samplesamong class C1 to C2. In the cases of there are multiple top scoringpairs (all sharing the same Dij), the top pair by a secondary rank scorethat measures the magnitude to which inversions of gene expressionlevels occur from one class to the other within a pair of genes isselected. The top pair with highest frequency of absolute Dij>2 fold inall samples will be selected as candidate pair. The candidate pair canthen be assessed in an independent testing data set. Leave-one-out crossvalidation (LOOCV) can be carried out in the training data set toevaluate how the algorithm perform. The performances of the predictorscan be assessed based on maximum misclassification error rate. All thestatistical analyses can be done using R (R Development Core Team,2006).

A review of the methods and statistic tools useful for determining areference level can be found in James Westgard, Ph.D., Basic MethodsValidation, 3d edition (2008), which is hereby incorporated by referencein its entirety. Specific references are made to Chapter 9 (“How isreportable range of a method determined”) and Chapter 15 (“How is areference interval verified”).

Clinically reportable range (CRR) is the range of analyte values that amethod can measure, allowing for specimen dilution, concentration, orother pretreatment used to extend the direct analytical measurementrange. As provided in the Basic Methods Validation by Dr. Westgard, theexperiment to be performed is often called a “linearity experiment,”though there technically is no requirement that a method provide alinear response unless two-point calibration is being used. This rangecan also be referred as the “linear range,” “analytical range,” or“working range” for a method.

The reportable range is assessed by inspection of the linearity graph.That inspection can involve manually drawing the best straight linethrough the linear portion of the points, drawing a point-to-point linethrough all the points then comparing with the best straight line, orfitting a regression line through the points in the linear range. Thereare more complicated statistical calculations that are recommended insome guidelines, such as Clinical Laboratory Standards Institute(CLSI)'s EP-6 protocol for evaluating the linearity of analyticalmethods. But it is commonly accepted that the reportable range can beadequately determined from a “visual” assessment, i.e., by manuallydrawing the best straight line that fits the lowest points in theseries. The Clinical Laboratory Standards Institute (CLSI) recommends aminimum of at least 4-preferably 5-different levels of concentrations.More than 5 can be used, particularly if the upper limit of reportablerange needs to be maximized, but 5 levels are convenient and almostalways sufficient.

A reference interval is typically established by assaying specimens thatare obtained from individuals that meet carefully defined criteria(reference sample group). Protocols such as those of the InternationalFederation of Clinical Chemistry (IFCC) Expert Panel on Theory ofReference Values and the CLSI delineate comprehensive systematicprocesses that use carefully selected reference sample groups toestablish reference intervals. These protocols typically need a minimumof 120 reference individuals for each group (or subgroup) that needs tobe characterized.

The CLSI Approved Guideline C28-A2 describes different ways for alaboratory to validate the transference of established referenceintervals to the individual laboratory that includes 1. Divine judgment,wherein the laboratory simply reviews the information submitted andsubjectively verifies that the reference intervals are applicable to theadopting laboratory's patient population and test methods; 2.Verification with 20 samples, wherein experimental validation isperformed by collecting and analyzing specimens from 20 individuals whorepresent the reference sample population; 3. Estimation with 60samples, wherein an experimental validation is performed by collectingand analyzing specimens from 60 individuals who represent the referencesample population, and the actual reference interval is estimated andcompared to the claimed or reported interval using a statistical formulacomparing the means and standard deviations of the two populations; and4. Calculation from comparative method, wherein one can adjust orcorrect the claimed or reported reference intervals on the basis of theobserved methodological bias and the mathematical relationshipdemonstrated between the analytical methods being used.

A person of ordinary skill in the art would understand that thereference expression level of the biomarkers disclosed herein as well asthe reference ratios between two biomarkers can be determined by one ormore methods as provided herein or other methods known in the art.

5. Mutant HRAS as a Biomarker for FTI Treatment 5.1. HRAS MutationStatus

The HRAS protein is involved in regulating cell division in response togrowth factor stimulation. Growth factors act by binding cell surfacereceptors that span the cell's plasma membrane. Once activated,receptors stimulate signal transduction events in the cytoplasm, aprocess by which proteins and second messengers relay signals fromoutside the cell to the cell nucleus and instruct the cell to grow ordivide. HRAS is localized in the plasma membrane, and is an early playerin many signal transduction pathways. HRAS acts as a molecular on/offswitch—once it is turned on it recruits and activates proteins necessaryfor the propagation of the receptor's signal. In certain tumors,mutations in HRAS or its upstream effectors cause it to be permanentlyon, resulting in persistent activation of downstream growth andproliferation signals that drive tumor cell growth. FTIs work to preventthe aberrant growth and proliferation of cells that are dependent onthese signaling pathways by inhibiting protein farnesylation andsubsequent membrane localization of HRAS, thereby switching HRAS off.

FTIs such as tipifarnib prevent protein farnesylation, a type of proteinmodification known as prenylation, which along with other proteinmodifications, allows membrane localization of HRAS where it can receiveand transmit extracellular signals implicated in cancer initiation anddevelopment. FTIs such as tipifarnib can block HRAS farnesylation andsubsequent membrane localization, and inhibit oncogenic, HRAS-drivencellular transformation in vitro and in vivo. While K-ras and N-rassimilarly utilize protein farnesylation, they can also utilize a relatedprenylation pathway that also leads to membrane localization. Meanwhile,HRAS membrane localization is solely dependent on protein farnesylation.

The head and neck cancers and the lung cancers to be treated by methodsprovided herein have HRAS mutations. Methods provided herein orotherwise known in the art can be used to determine the mutation statusof an HRAS gene. In some embodiments, the mutation status of an HRASgene can be determined an NGS-based assay. In some embodiments, themutation status of an HRAS gene can be determined by a qualitativePCR-based assay. A qualitative PCR based assay can be technicallysimilar to the PCR-based assays already developed and approved by theFDA for K-ras. In some embodiments, mutation status of an HRAS gene canbe determined in the form of a companion diagnostic to the FTItreatment, such as the tipifarnib treatment. The companion diagnosticcan be performed at the clinic site where the patient receives thetipifarnib treatment, or at a separate site.

Provided herein are methods of treating EGFR inhibitor-refractorysquamous cell carcinoma of the head and neck (SCCHN), wherein the SCCHNhas an HRAS mutation, comprising administering to the subject afarnesyltransferase inhibitor (FTI). In certain embodiments, said HRASmutation comprises an amino acid substitution at a codon selected from agroup consisting of G12, G13, Q61, Q22, K117, A146 and any combinationthereof. In certain embodiments, said SCCHN does not have K-Ras mutationor N-Ras mutation. In certain embodiments, said SCCHN has wild typeK-Ras and wild type N-Ras. In certain embodiments, said SCCHN is HPVnegative. In certain embodiments, said SCCHN is HPV positive. In certainembodiments, said SCCHN is at an advanced stage or metastatic. Incertain embodiments, said SCCHN is relapsed SCCHN. In specificembodiments, the SCCHN is SCCHN of the trachea. In specific embodiments,the SCCHN is SCCHN of the maxilla. In specific embodiments, the SCCHN isSCCHN of the oral cavity. In certain embodiments, the EGFR inhibitor iscetuximab. In certain embodiments, the EGFR inhibitor is erlotinib. Incertain embodiments, the EGFR inhibitor is gefitinib. In certainembodiments, the EGFR inhibitor is panitumumab. In certain embodiments,the FTI is tipifarnib. In certain embodiments, said FTI is administeredin combination with chemotherapy. In certain embodiments, saidchemotherapy comprises a platinum-based therapy, a taxane, or acombination thereof.

Provided herein are methods to treat SCCHN in a subject with an FTI orselecting SCCHN patients for an FTI treatment based on the presence of aHRAS mutation, wherein the SCCHN is refractory to treatment with an EGFRinhibitor. In some embodiments, the SCCHN is HPV negative. In someembodiments, said SCCHN is HPV positive. In some embodiments, themethods include (a) determining the SCCHN to be refractory to treatmentwith an EGFR inhibitor, (b) determining the SCCHN patient to have a HRASmutation, and subsequently (c) administering a therapeutically effectiveamount of tipifarnib to the patient. In some embodiments, providedherein are methods of treating an EGFR-inhibitor-resistant squamous cellcarcinoma of the head and neck in a subject with an FTI. In someembodiments, the EGFR inhibitor is cetuximab. In certain embodiments,the EGFR inhibitor is erlotinib. In certain embodiments, the EGFRinhibitor is gefitinib. In certain embodiments, the EGFR inhibitor ispanitumumab. In some embodiments, the EGFR inhibitor is panitumumab. Incertain embodiments, said tipifarnib is administered in combination withchemotherapy. In certain embodiments, said chemotherapy comprises aplatinum-based therapy, a taxane, or a combination thereof.

In some embodiments, provided herein are methods to treat SCCHN in asubject with an FTI or selecting SCCHN patients for an FTI treatmentbased on the presence of a HRAS mutation, wherein the patient has neverbeen treated with an EGFR inhibitor. In some embodiments, the SCCHN isHPV negative. In some embodiments, said SCCHN is HPV positive. In someembodiments, the methods include (a) determining that the patient hasnever been treated with an EGFR inhibitor, (b) determining the SCCHNpatient to have a HRAS mutation, and subsequently (c) administering atherapeutically effective amount of tipifarnib to the patient and notadministering an EGFR inhibitor. In some embodiments, the EGFR inhibitoris cetuximab. In certain embodiments, the EGFR inhibitor is erlotinib.In certain embodiments, the EGFR inhibitor is gefitinib. In certainembodiments, the EGFR inhibitor is panitumumab. In some embodiments, theEGFR inhibitor is panitumumab. In certain embodiments, said tipifarnibis administered in combination with chemotherapy. In certainembodiments, said chemotherapy comprises a platinum-based therapy, ataxane, or a combination thereof.

Provided herein are methods of treating EGFR inhibitor-refractory lungsquamous cell carcinoma (lung SCC), wherein the lung SCC has an HRASmutation, comprising administering to the subject a farnesyltransferaseinhibitor (FTI). In certain embodiments, said HRAS mutation comprises anamino acid substitution at a codon selected from a group consisting ofG12, G13, Q61, Q22, K117, A146 and any combination thereof. In certainembodiments, said lung SCC does not have K-Ras mutation or N-Rasmutation. In certain embodiments, said lung SCC has wild type K-Ras andwild type N-Ras. In certain embodiments, said lung SCC is HPV negative.In certain embodiments, said lung SCC is HPV positive. In certainembodiments, said lung SCC is at an advanced stage or metastatic. Incertain embodiments, said lung SCC is relapsed lung SCC. In certainembodiments, the EGFR inhibitor is cetuximab. In certain embodiments,the EGFR inhibitor is erlotinib. In certain embodiments, the EGFRinhibitor is gefitinib. In certain embodiments, the EGFR inhibitor ispanitumumab. In certain embodiments, the FTI is tipifarnib. In certainembodiments, said FTI is administered in combination with chemotherapy.In certain embodiments, said chemotherapy comprises a platinum-basedtherapy, a taxane, or a combination thereof.

Provided herein are methods to treat lung SCC in a subject with an FTIor selecting lung SCC patients for an FTI treatment based on thepresence of a HRAS mutation, wherein the lung SCC is refractory totreatment with an EGFR inhibitor. In some embodiments, the lung SCC isHPV negative. In some embodiments, said lung SCC is HPV positive. Insome embodiments, the methods include (a) determining the lung SCC to berefractory to treatment with an EGFR inhibitor, (b) determining the lungSCC patient to have a HRAS mutation, and subsequently (c) administeringa therapeutically effective amount of tipifarnib to the patient. In someembodiments, provided herein are methods of treating anEGFR-inhibitor-resistant lung squamous cell carcinoma in a subject withan FTI. In some embodiments, the EGFR inhibitor is cetuximab. In certainembodiments, the EGFR inhibitor is erlotinib. In certain embodiments,the EGFR inhibitor is gefitinib. In certain embodiments, the EGFRinhibitor is panitumumab. In some embodiments, the EGFR inhibitor ispanitumumab. In certain embodiments, said tipifarnib is administered incombination with chemotherapy. In certain embodiments, said chemotherapycomprises a platinum-based therapy, a taxane, or a combination thereof.

In some embodiments, provided herein are methods to treat lung SCC in asubject with an FTI or selecting lung SCC patients for an FTI treatmentbased on the presence of a HRAS mutation, wherein the patient has neverbeen treated with an EGFR inhibitor. In some embodiments, the lung SCCis HPV negative. In some embodiments, said lung SCC is HPV positive. Insome embodiments, the methods include (a) determining that the patienthas never been treated with an EGFR inhibitor, (b) determining the lungSCC patient to have a HRAS mutation, and subsequently (c) administeringa therapeutically effective amount of tipifarnib to the patient and notadministering an EGFR inhibitor. In some embodiments, the EGFR inhibitoris cetuximab. In certain embodiments, the EGFR inhibitor is erlotinib.In certain embodiments, the EGFR inhibitor is gefitinib. In certainembodiments, the EGFR inhibitor is panitumumab. In some embodiments, theEGFR inhibitor is panitumumab. In certain embodiments, said tipifarnibis administered in combination with chemotherapy. In certainembodiments, said chemotherapy comprises a platinum-based therapy, ataxane, or a combination thereof.

In some embodiments, the HRAS mutation is a mutation at a codon selectedfrom the group consisting of G12, G13, Q61, Q22, K117, and A146. In someembodiments, the HRAS mutation can be a mutation selected from the groupconsisting of the amino acid substitutions of G12R, G12V, G13C, G13R,Q61L, Q61R, Q22K, K117N, and A146P. In some embodiments, the mutationcan be a mutation at another codon that results in activation of HRASprotein.

In some embodiments, the methods provided herein further include (a)determining the presence or absence of a K-Ras mutation and a N-Rasmutation in a sample from the subject, and subsequently (b)administering a therapeutically effective amount of an FTI to thesubject if the sample does not have the K-Ras mutation or the N-Rasmutation. In some embodiments, the method includes administering atherapeutically effective amount of an FTI to the subject if the samplehas wild type K-Ras and wild type N-Ras. In certain embodiments, saidFTI is administered in combination with chemotherapy. In certainembodiments, said chemotherapy comprises a platinum-based therapy, ataxane, or a combination thereof.

In some embodiments, the K-Ras mutation is KA-Ras mutation. In someembodiments, the K-Ras mutation is KB-Ras mutation. In some embodiments,the K-Ras mutation is a combination of KA-Ras mutation and a KB-Rasmutation. The K-Ras mutation can include a mutation at a codon selectedfrom the group consisting of G12, G13, and Q61 of KA-Ras, KB-Ras, orboth. In some embodiments, the KA-Ras mutation can include a mutationselected from the group consisting of the amino acid substitutions G12C,G12D, G12A, G12V, G12S, G12F, G12R, G12N, G13C, G13D, G13R, G13S, G13N,Q61 K, Q61 H, Q61 L, Q61 P, Q61 R and A146V. In some embodiments, theKB-Ras mutation can include a mutation selected from the groupconsisting of the amino acid substitutions G12C, G12D, G12A, G12V, G12S,G12F, G12R, G12N, G13C, G13D, G13R, G13S, G13N, Q61 K, Q61 H, Q61 L, Q61P, Q61 R and A146V.

In some embodiments, the N-Ras mutation can include at least onemutation at a codon selected from the group consisting of G12, G13, G15,G60 and Q61. In some embodiments, the N-Ras mutation can include atleast one mutation at a codon selected from the group consisting of G12,G13, and Q61. In some embodiments, the N-Ras mutation can include atleast one mutation selected from the group consisting of the amino acidsubstitutions of G12C, G12D, G12F, G12S, G12A, G12V, G12R, G13C, G13R,G13A, G13D, G13V, G15W, G60E, Q61P, Q61L, Q61R, Q61K, Q61H and Q61E.

In some embodiments, the sample is determined to not have amino acidsubstitution at G12, G13, and Q61 of K-Ras, and also not have amino acidsubstitution at G12, G13, and Q61 of N-Ras. In some embodiments, thesample is determined to not have any K-Ras mutation or any N-Rasmutation. In some embodiments, the sample is determined to have wildtype K-Ras and wild type N-Ras.

In some embodiments, the method provided herein includes (a) determiningthe presence or absence of a HRAS mutation, a K-Ras mutation, and aN-Ras mutation in a sample from the subject, and subsequently (b)administering a therapeutically effective amount of an FTI to thesubject if the sample is determined to have a HRAS mutation, but noK-Ras mutation or N-Ras mutation. The sample can be a tumor sample. Insome embodiments, the methods include (a) determining the SCCHN patientto have a HRAS mutation and wild type K-Ras and wild type N-Ras, andsubsequently (b) administering a therapeutically effective amount of anFTI to the subject. In some embodiments, the FTI is tipifarnib. Incertain embodiments, said FTI is administered in combination withchemotherapy. In certain embodiments, said chemotherapy comprises aplatinum-based therapy, a taxane, or a combination thereof.

Provided herein are methods to treat the SCCHN in a subject with an FTI,and methods for selecting cancer patients for an FTI treatment based onthe presence of a HRAS mutation. Provided herein are also methods totreat a premalignant head and neck condition in a subject with an FTI,and methods for selecting patients with a premalignant head and neckcondition for an FTI treatment based on HRAS mutation status.

In some embodiments, provided herein are methods to treat SCCHN in asubject with an FTI or selecting SCCHN patients for an FTI treatmentbased on the presence of a HRAS mutation. The cancer can be related toHuman papillomavirus (HPV+ or HPV positive), or unrelated to HPV (HPV-or HPV negative).

Provided herein are methods for predicting responsiveness of SCCHNpatient to an FTI treatment, methods for SCCHN patient populationselection for an FTI treatment, and methods for treating SCCHN in asubject with a therapeutically effective amount of an FTI, based on thepresence of a HRAS mutation in a sample from the patient. The mutationstatus of HRAS can be detected at the nucleic acid or protein level. Insome embodiments, the HRAS mutation status is determined by analyzingnucleic acids obtained from the sample. In some embodiments, the HRASmutation status is determined by analyzing protein obtained from thesample.

In some embodiments, the methods include (a) determining the lung SCCpatient to have a HRAS mutation and wild type K-Ras and wild type N-Ras,and subsequently (b) administering a therapeutically effective amount ofan FTI to the subject. In some embodiments, the FTI is tipifarnib. Incertain embodiments, said FTI is administered in combination withchemotherapy. In certain embodiments, said chemotherapy comprises aplatinum-based therapy, a taxane, or a combination thereof.

Provided herein are methods to treat the lung SCC in a subject with anFTI, and methods for selecting cancer patients for an FTI treatmentbased on the presence of a HRAS mutation. Provided herein are alsomethods to treat a premalignant lung condition in a subject with an FTI,and methods for selecting patients with a premalignant lung conditionfor an FTI treatment based on HRAS mutation status.

In some embodiments, provided herein are methods to treat lung SCC in asubject with an FTI or selecting lung SCC patients for an FTI treatmentbased on the presence of a HRAS mutation. The cancer can be related toHuman papillomavirus (HPV+ or HPV positive), or unrelated to HPV (HPV-or HPV negative).

Provided herein are methods for predicting responsiveness of lung SCCpatient to an FTI treatment, methods for lung SCC patient populationselection for an FTI treatment, and methods for treating lung SCC in asubject with a therapeutically effective amount of an FTI, based on thepresence of a HRAS mutation in a sample from the patient. The mutationstatus of HRAS can be detected at the nucleic acid or protein level. Insome embodiments, the HRAS mutation status is determined by analyzingnucleic acids obtained from the sample. In some embodiments, the HRASmutation status is determined by analyzing protein obtained from thesample.

In some embodiments, the HRAS mutation status is determined by analyzingnucleic acids obtained from the sample. The nucleic acids may be mRNA orgenomic DNA molecules from the test subject. Methods for determining Rasmutation status by analyzing nucleic acids are well known in the art. Insome embodiments, the methods include sequencing, Polymerase ChainReaction (PCR), DNA microarray, Mass Spectrometry (MS), SingleNucleotide Polymorphism (SNP) assay, denaturing high-performance liquidchromatography (DHPLC), or Restriction Fragment Length Polymorphism(RFLP) assay. In some embodiments, the Ras mutation status is determinedusing standard sequencing methods, including, for example, Sangersequencing, next generation sequencing (NGS). In some embodiments, theRas mutation status is determined using MS.

In some embodiments, the HRAS mutation status is determined by analyzingprotein obtained from the sample. The mutated Ras H-protein can bedetected by a variety of immunohistochemistry (IHC) approaches,Immunoblotting assay, Enzyme-Linked Immunosorbent Assay (ELISA) or otherimmunoassay methods known in the art.

As a person of ordinary skill in the art would understand, any methodsdescribed herein or otherwise known in the art for analyzing Rasmutation can be used to determining the presence or absence of a HRASmutation.

5.2. Samples

In some embodiments, methods provided herein include obtaining a samplefrom the subject. In some embodiments, the sample is a tumor sample. Insome embodiments, the sample used in the present methods includes abiopsy (e.g., a tumor biopsy). The biopsy can be from any organ ortissue, for example, skin, liver, lung, heart, colon, kidney, bonemarrow, teeth, lymph node, hair, spleen, brain, breast, or other organs.Any biopsy technique known by those skilled in the art can be used forisolating a sample from a subject, for instance, open biopsy, closebiopsy, core biopsy, incisional biopsy, excisional biopsy, or fineneedle aspiration biopsy.

The sample used in the methods provided herein includes body fluids froma subject. Non-limiting examples of body fluids include blood (e.g.,peripheral whole blood, peripheral blood), blood plasma, bone marrow,amniotic fluid, aqueous humor, bile, lymph, menses, serum, urine,cerebrospinal fluid surrounding the brain and the spinal cord, synovialfluid surrounding bone joints.

In one embodiment, the sample is a bone marrow sample. Procedures toobtain a bone marrow sample are well known in the art, including but notlimited to bone marrow biopsy and bone marrow aspiration. Bone marrowhas a fluid portion and a more solid portion. In bone marrow biopsy, asample of the solid portion is taken. In bone marrow aspiration, asample of the fluid portion is taken. Bone marrow biopsy and bone marrowaspiration can be done at the same time and referred to as a bone marrowexam.

In some embodiments, the sample is a blood sample. The blood sample canbe obtained using conventional techniques as described in, e.g. Innis etal, editors, PCR Protocols (Academic Press, 1990). White blood cells canbe separated from blood samples using convention techniques orcommercially available kits, e.g. RosetteSep kit (Stein CellTechnologies, Vancouver, Canada). Sub-populations of white blood cells,e.g. mononuclear cells, NK cells, B cells, T cells, monocytes,granulocytes or lymphocytes, can be further isolated using conventionaltechniques, e.g. magnetically activated cell sorting (MACS) (MiltenyiBiotec, Auburn, Calif.) or fluorescently activated cell sorting (FACS)(Becton Dickinson, San Jose, Calif.).

In certain embodiments, the sample used in the methods provided hereinincludes a plurality of cells. Such cells can include any type of cells,e.g., stem cells, blood cells (e.g., PBMCs), lymphocytes, NK cells, Bcells, T cells, monocytes, granulocytes, immune cells, or tumor orcancer cells. Specific cell populations can be obtained using acombination of commercially available antibodies (e.g., Quest Diagnostic(San Juan Capistrano, Calif.); Dako (Denmark)).

In certain embodiments, the sample used in the methods provided hereinis from a diseased tissue, e.g., from an individual having SCCHN. Insome embodiments, the cells can be obtained from the tumor or cancercells or a tumor tissue, such as a tumor biopsy or a tumor explants. Incertain embodiments, the number of cells used in the methods providedherein can range from a single cell to about 10⁹ cells. In someembodiments, the number of cells used in the methods provided herein isabout 1×10⁴, 5×10⁴, 1×10⁵, 5×10⁵, 1×10⁶, 5×10⁶, 1×10⁷, 5×10⁷, 1×10⁸, or5×10⁸.

The number and type of cells collected from a subject can be monitored,for example, by measuring changes in morphology and cell surface markersusing standard cell detection techniques such as flow cytometry, cellsorting, immunocytochemistry (e.g., staining with tissue specific orcell-marker specific antibodies) fluorescence activated cell sorting(FACS), magnetic activated cell sorting (MACS), by examination of themorphology of cells using light or confocal microscopy, and/or bymeasuring changes in gene expression using techniques well known in theart, such as PCR and gene expression profiling. These techniques can beused, too, to identify cells that are positive for one or moreparticular markers. Fluorescence activated cell sorting (FACS) is awell-known method for separating particles, including cells, based onthe fluorescent properties of the particles (Kamarch, 1987, MethodsEnzymol, 151:150-165). Laser excitation of fluorescent moieties in theindividual particles results in a small electrical charge allowingelectromagnetic separation of positive and negative particles from amixture. In one embodiment, cell surface marker-specific antibodies orligands are labeled with distinct fluorescent labels. Cells areprocessed through the cell sorter, allowing separation of cells based ontheir ability to bind to the antibodies used. FACS sorted particles maybe directly deposited into individual wells of 96-well or 384-wellplates to facilitate separation and cloning.

In certain embodiments, subsets of cells are used in the methodsprovided herein. Methods to sort and isolate specific populations ofcells are well-known in the art and can be based on cell size,morphology, or intracellular or extracellular markers. Such methodsinclude, but are not limited to, flow cytometry, flow sorting, FACS,bead based separation such as magnetic cell sorting, size-basedseparation (e.g., a sieve, an array of obstacles, or a filter), sortingin a microfluidics device, antibody-based separation, sedimentation,affinity adsorption, affinity extraction, density gradientcentrifugation, laser capture microdissection, etc.

5.3. Cancers

Provided herein are methods of treating EGFR inhibitor-refractorysquamous cell carcinoma of the head and neck (SCCHN), wherein the SCCHNhas an HRAS mutation, comprising administering to the subject afarnesyltransferase inhibitor (FTI). In certain embodiments, said HRASmutation comprises an amino acid substitution at a codon selected from agroup consisting of G12, G13, Q61, Q22, K117, A146, and any combinationthereof. In certain embodiments, said SCCHN does not have K-Ras mutationor N-Ras mutation. In certain embodiments, said SCCHN has wild typeK-Ras and wild type N-Ras. In certain embodiments, said SCCHN is HPVnegative. In certain embodiments, said SCCHN is HPV positive. In certainembodiments, said SCCHN is at an advanced stage or metastatic. Incertain embodiments, said SCCHN is relapsed SCCHN. In specificembodiments, the SCCHN is SCCHN of the trachea. In specific embodiments,the SCCHN is SCCHN of the maxilla. In specific embodiments, the SCCHN isSCCHN of the oral cavity. In certain embodiments, the EGFR inhibitor iscetuximab. In certain embodiments, the EGFR inhibitor is erlotinib. Incertain embodiments, the EGFR inhibitor is gefitinib. In certainembodiments, the EGFR inhibitor is panitumumab. In certain embodiments,the FTI is tipifarnib. In certain embodiments, said FTI is administeredin combination with chemotherapy. In certain embodiments, saidchemotherapy comprises a platinum-based therapy, a taxane, or acombination thereof.

In some embodiments, provided herein are methods of treating EGFRinhibitor-refractory SCCHN in a subject having an HRAS mutation,comprising administering to the subject an FTI. In certain embodiments,said HRAS mutation comprises an amino acid substitution at a codonselected from a group consisting of G12, G13, Q61, Q22, K117, A146, andany combination thereof. In certain embodiments, said SCCHN does nothave K-Ras mutation or N-Ras mutation. In certain embodiments, saidSCCHN has wild type K-Ras and wild type N-Ras. In certain embodiments,said SCCHN is HPV negative. In certain embodiments, said SCCHN is HPVpositive. In certain embodiments, said SCCHN is at an advanced stage ormetastatic. In certain embodiments, said SCCHN is relapsed SCCHN. Inspecific embodiments, the SCCHN is SCCHN of the trachea. In specificembodiments, the SCCHN is SCCHN of the maxilla. In specific embodiments,the SCCHN is SCCHN of the oral cavity. In certain embodiments, the EGFRinhibitor is cetuximab. In certain embodiments, the EGFR inhibitor iserlotinib. In certain embodiments, the EGFR inhibitor is gefitinib. Incertain embodiments, the EGFR inhibitor is panitumumab. In certainembodiments, the FTI is tipifarnib. In certain embodiments, said FTI isadministered in combination with chemotherapy. In certain embodiments,said chemotherapy comprises a platinum-based therapy, a taxane, or acombination thereof.

In some embodiments, provided herein are methods to treat SCCHN in asubject with an FTI or selecting SCCHN patients for an FTI treatmentbased on the presence of a HRAS mutation. In some embodiments, the SCCHNis HPV negative. In some embodiments, said SCCHN is HPV positive. Insome embodiments, the methods include (a) determining a HPV negativeSCCHN patient to have a HRAS mutation, and subsequently (b)administering a therapeutically effective amount of tipifarnib to thepatient. In certain embodiments, said tipifarnib is administered incombination with chemotherapy. In certain embodiments, said chemotherapycomprises a platinum-based therapy, a taxane, or a combination thereof.

In some embodiments, provided herein are methods to treat SCCHN in asubject with an FTI or selecting SCCHN patients for an FTI treatmentbased on the presence of a HRAS mutation and resistance to an EGFRinhibitor. In some embodiments, the SCCHN is HPV negative. In someembodiments, said SCCHN is HPV positive. In some embodiments, themethods include (a) determining a HPV negative SCCHN patient to have aHRAS mutation and be resistant to an EGFR inhibitor, and subsequently(b) administering a therapeutically effective amount of tipifarnib tothe patient. In certain embodiments, said tipifarnib is administered incombination with chemotherapy. In certain embodiments, said chemotherapycomprises a platinum-based therapy, a taxane, or a combination thereof.

Head and neck squamous cell carcinoma (SCCHN) is the 6^(th) most commoncancer worldwide, with about 650,000 cases and 200,000 deaths per yearworldwide, and about 54,000 new cases per year in the US. It is also themost common cancer in central Asia.

SCCHN has 2 different etiologies and corresponding tumor types. Thefirst subtype is associated with tobacco smoking and alcoholconsumption, and unrelated to Human papillomavirus (HPV− or HPVnegative). The second subtype is associated with infection withhigh-risk HPV (HPV+ or HPV positive). The second subtype is largelylimited to oropharyngeal cancers. HPV+ tumors are distinct entity withbetter prognosis and may require differential treatments.

A significant proportion of SCCHN, particularly oropharyngeal cancers,are caused by HPV infection. High-risk HPV subtype 16 accounts for 85%of all HPV+ tumors in SCCHN. P16 can be used as surrogate marker of HPVinfection in SCCHN, particularly in the oropharynx. More accurate HPVtesting is available and based on E6/E7 detection (Liang C, et al.Cancer Res. 2012; 72:5004-5013).

HPV+ SCCHN show significantly lower EGFR expression levels thanHPV-SCCHN. EGFR amplification only occurs in HPV-SCCHN. High EGFR genecopy number and protein expression are associated with poor clinicaloutcome in advanced SCCHN.

Currently, first-line therapy for recurrent/metastatic SCCHN includeplatinum-based doublet (e.g., cisplatin/5-FU or carboplatin/paclitaxel),optionally in combination with anti-EGFR antibody therapy (e.g.Cetuximab, Panitumumab, Afatinib). Second-line therapy includes taxanes,methotrexate, and/or cetuximab. Anti-EGFR antibody therapy, such asCetuximab (a chimeric IgG1) or Panitumumab can be used as a singleagent, with chemotherapy (e.g. Platinum/5-FU, Cisplatin), or withradiation therapy. Despite high EGFR expression levels in SCCHN,single-agent response rate for Cetuximab is only 13% with SD rate of33%, and there is currently no predictive biomarker available.

Drugs in development for SCCHN include those targeting PI3K pathway:BKM120 (buparlisib)+cetuximab, BYL719+cetuximab, Temsirolimus+cetuximab,Rigosertib+cetuximab; those targeting MET pathway: Tivantinib+cetuximab,Ficlatuzumab+cetuximab; those targeting EGFR/HER3 pathwayAfatinib+cetuximab±paclitaxel, Patritumab; those targeting FGFR pathway:BGJ398; those targeting CDK4/6-cell cycle pathway: Palbociclib, LEE011;RTK inhibitor: Anlotinib and chemotherapy: Oral Azacitidine. More recenttherapeutic options for SCCHN include immunotherapy, such as anti-PD1 oranti-PDL1 antibodies.

While high cure rates have been achieved for localized and loco-regionaldisease using surgery, radiation, chemoradiation, and inductionchemotherapy, survival rates for recurrent/metastatic diseases remainvery poor, and better treatment options are necessary.

In some embodiments, provided herein are methods to treat SCCHN in asubject with an FTI or selecting SCCHN patients for an FTI treatmentbased on the presence of a HRAS mutation, wherein the SCCHN isrefractory to treatment with an EGFR inhibitor. In some embodiments, theSCCHN is HPV negative. In some embodiments, said SCCHN is HPV positive.In some embodiments, the methods include (a) determining SCCHN to berefractory to treatment with an EGFR inhibitor, (b) determining theSCCHN patient to have a HRAS mutation, and subsequently (c)administering a therapeutically effective amount of tipifarnib to thepatient. In some embodiments, provided herein are methods of treating anEGFR-inhibitor-resistant squamous cell carcinoma in a subject with anFTI. In some embodiments, the EGFR inhibitor is cetuximab. In someembodiments, the EGFR inhibitor is erlotinib. In some embodiments, theEGFR inhibitor is gefitinib. In some embodiments, the EGFR inhibitor ispanitumumab. In some embodiments, the EGFR inhibitor is panitumumab. Insome embodiments, the patient has been treated with an EGFR inhibitor,which resulted in progressive disease. In certain embodiments, saidtipifarnib is administered in combination with chemotherapy. In certainembodiments, said chemotherapy comprises a platinum-based therapy, ataxane, or a combination thereof.

In some embodiments, provided herein are methods to treat SCCHN in asubject with an FTI or selecting SCCHN patients for an FTI treatmentbased on the presence of a HRAS mutation, wherein the patient has neverbeen treated with an EGFR inhibitor. In some embodiments, the SCCHN isHPV negative. In some embodiments, said SCCHN is HPV positive. In someembodiments, the methods include (a) determining that the patient hasnever been treated with an EGFR inhibitor, (b) determining the SCCHNpatient to have a HRAS mutation, and subsequently (c) administering atherapeutically effective amount of tipifarnib to the patient and notadministering an EGFR inhibitor. In some embodiments, the EGFR inhibitoris cetuximab. In some embodiments, the EGFR inhibitor is erlotinib. Insome embodiments, the EGFR inhibitor is gefitinib. In some embodiments,the EGFR inhibitor is panitumumab. In some embodiments, the EGFRinhibitor is panitumumab. In certain embodiments, said tipifarnib isadministered in combination with chemotherapy. In certain embodiments,said chemotherapy comprises a platinum-based therapy, a taxane, or acombination thereof.

The EGFR inhibitor to which the SCCHN is refractory may be any EGFRinhibitor known in the art. The EGFR inhibitor may be an anti-EGFRantibody or antigen-binding fragment thereof, for example, cetuximab(ERBITUX®; Bristol-Myers Squibb/Lilly), panitumumab (VECTIBIX®; Amgen),or zalutumumab (Genmab). The EGFR inhibitor may also be a small moleculeinhibitor. Examples of EGFR inhibitors include, but are not limited to,reversible and irreversible inhibitors, such as erlotinib (TARCEVA®;Genentech/Astellas Oncology), AZD9291 (AstraZeneca), gefitinib (IRESSA®;AstraZeneca), icotinib (BPI-2009H; Beta Pharma), rociletinib (CO-1686,AVL-301; Clovis Oncology), poziotinib (NOV120101, HM781-36B; HanmiPharmaceuticals/Spectrum Pharmaceuticals), afatinib (BIBW2292;Boehringer Ingelheim), pelitinib (EKB-569; Wyeth Pharmaceuticals),ASP8273 (Astellas), Luminespib (AUY922; Vernalis/Novartis), and XL647(Exelixis).

In some embodiments, provided herein is a method of treating SCCHN in asubject based on the presence of a HRAS mutation and resistance to anEGFR inhibitor. In some embodiments, the SCCHN can be HPV negativeSCCHN. In some embodiments, the SCCHN can be HPV positive SCCHN. In someembodiments, the SCCHN can be relapsed/recurrent SCCHN. In someembodiments, the SCCHN can be metastatic SCCHN. The method providedherein includes (a) determining the presence or absence of a HRASmutation in a sample from the subject, and subsequently (b)administering a therapeutically effective amount of an FTI to thesubject if the sample is determined to have a HRAS mutation. The methodprovided herein includes (a) determining the presence or absence of aHRAS mutation in a sample from the subject, (b) determining resistanceto an EGFR inhibitor, and subsequently (c) administering atherapeutically effective amount of an FTI to the subject if the sampleis determined to have a HRAS mutation and be resistant to an EGFRinhibitor. The sample can be a tumor sample. In some embodiments, themethods include (a) determining a SCCHN patient to have a HRAS mutation,and subsequently (b) administering a therapeutically effective amount ofan FTI to the subject. In some embodiments, the methods include (a)determining a SCCHN patient to have a HRAS mutation, and (b) determiningresistance to an EGFR inhibitor, and, subsequently (c) administering atherapeutically effective amount of an FTI to the subject. In someembodiments, the FTI is tipifarnib. In certain embodiments, said FTI isadministered in combination with chemotherapy. In certain embodiments,said chemotherapy comprises a platinum-based therapy, a taxane, or acombination thereof.

Provided herein are methods of treating EGFR inhibitor-refractory lungsquamous cell carcinoma (lung SCC), wherein the lung SCC has an HRASmutation, comprising administering to the subject a farnesyltransferaseinhibitor (FTI). In certain embodiments, said HRAS mutation comprises anamino acid substitution at a codon selected from a group consisting ofG12, G13, Q61, Q22, K117, A146, and any combination thereof. In certainembodiments, said lung SCC does not have K-Ras mutation or N-Rasmutation. In certain embodiments, said lung SCC has wild type K-Ras andwild type N-Ras. In certain embodiments, said lung SCC is HPV negative.In certain embodiments, said lung SCC is HPV positive. In certainembodiments, said lung SCC is at an advanced stage or metastatic. Incertain embodiments, said lung SCC is relapsed lung SCC. In certainembodiments, the EGFR inhibitor is cetuximab. In certain embodiments,the EGFR inhibitor is erlotinib. In certain embodiments, the EGFRinhibitor is gefitinib. In certain embodiments, the EGFR inhibitor ispanitumumab. In certain embodiments, the FTI is tipifarnib. In certainembodiments, said FTI is administered in combination with chemotherapy.In certain embodiments, said chemotherapy comprises a platinum-basedtherapy, a taxane, or a combination thereof.

In some embodiments, provided herein are methods of treating EGFRinhibitor-refractory lung SCC in a subject having an HRAS mutation,comprising administering to the subject an FTI. In certain embodiments,said HRAS mutation comprises an amino acid substitution at a codonselected from a group consisting of G12, G13, Q61, Q22, K117, A146, andany combination thereof. In certain embodiments, said lung SCC does nothave K-Ras mutation or N-Ras mutation. In certain embodiments, said lungSCC has wild type K-Ras and wild type N-Ras. In certain embodiments,said lung SCC is HPV negative. In certain embodiments, said lung SCC isHPV positive. In certain embodiments, said lung SCC is at an advancedstage or metastatic. In certain embodiments, said lung SCC is relapsedlung SCC. In certain embodiments, the EGFR inhibitor is cetuximab. Incertain embodiments, the EGFR inhibitor is erlotinib. In certainembodiments, the EGFR inhibitor is gefitinib. In certain embodiments,the EGFR inhibitor is panitumumab. In certain embodiments, the FTI istipifarnib. In certain embodiments, said FTI is administered incombination with chemotherapy. In certain embodiments, said chemotherapycomprises a platinum-based therapy, a taxane, or a combination thereof.

In some embodiments, provided herein are methods to treat lung SCC in asubject with an FTI or selecting lung SCC patients for an FTI treatmentbased on the presence of a HRAS mutation. In some embodiments, the lungSCC is HPV negative. In some embodiments, said lung SCC is HPV positive.In some embodiments, the methods include (a) determining a HPV negativelung SCC patient to have a HRAS mutation, and subsequently (b)administering a therapeutically effective amount of tipifarnib to thepatient. In certain embodiments, said tipifarnib is administered incombination with chemotherapy. In certain embodiments, said chemotherapycomprises a platinum-based therapy, a taxane, or a combination thereof.

In some embodiments, provided herein are methods to treat lung SCC in asubject with an FTI or selecting lung SCC patients for an FTI treatmentbased on the presence of a HRAS mutation and resistance to an EGFRinhibitor. In some embodiments, the lung SCC is HPV negative. In someembodiments, said lung SCC is HPV positive. In some embodiments, themethods include (a) determining a HPV negative lung SCC patient to havea HRAS mutation and be resistant to an EGFR inhibitor, and subsequently(b) administering a therapeutically effective amount of tipifarnib tothe patient. In certain embodiments, said tipifarnib is administered incombination with chemotherapy. In certain embodiments, said chemotherapycomprises a platinum-based therapy, a taxane, or a combination thereof.

In some embodiments, provided herein are methods to treat lung SCC in asubject with an FTI or selecting lung SCC patients for an FTI treatmentbased on the presence of a HRAS mutation, wherein the lung SCC isrefractory to treatment with an EGFR inhibitor. In some embodiments, thelung SCC is HPV negative. In some embodiments, said lung SCC is HPVpositive. In some embodiments, the methods include (a) determining lungSCC to be refractory to treatment with an EGFR inhibitor, (b)determining the lung SCC patient to have a HRAS mutation, andsubsequently (c) administering a therapeutically effective amount oftipifarnib to the patient. In some embodiments, provided herein aremethods of treating an EGFR-inhibitor-resistant squamous cell carcinomain a subject with an FTI. In some embodiments, the EGFR inhibitor iscetuximab. In some embodiments, the EGFR inhibitor is erlotinib. In someembodiments, the EGFR inhibitor is gefitinib. In some embodiments, theEGFR inhibitor is panitumumab. In some embodiments, the patient has beentreated with an EGFR inhibitor, which resulted in progressive disease.In certain embodiments, said tipifarnib is administered in combinationwith chemotherapy. In certain embodiments, said chemotherapy comprises aplatinum-based therapy, a taxane, or a combination thereof.

In some embodiments, provided herein are methods to treat lung SCC in asubject with an FTI or selecting lung SCC patients for an FTI treatmentbased on the presence of a HRAS mutation, wherein the patient has neverbeen treated with an EGFR inhibitor. In some embodiments, the lung SCCis HPV negative. In some embodiments, said lung SCC is HPV positive. Insome embodiments, the methods include (a) determining that the patienthas never been treated with an EGFR inhibitor, (b) determining the lungSCC patient to have a HRAS mutation, and subsequently (c) administeringa therapeutically effective amount of tipifarnib to the patient and notadministering an EGFR inhibitor. In some embodiments, the EGFR inhibitoris cetuximab. In some embodiments, the EGFR inhibitor is erlotinib. Insome embodiments, the EGFR inhibitor is gefitinib. In some embodiments,the EGFR inhibitor is panitumumab. In certain embodiments, saidtipifarnib is administered in combination with chemotherapy. In certainembodiments, said chemotherapy comprises a platinum-based therapy, ataxane, or a combination thereof.

The EGFR inhibitor to which the lung SCC is refractory may be any EGFRinhibitor known in the art. The EGFR inhibitor may be an anti-EGFRantibody or antigen-binding fragment thereof, for example, cetuximab(ERBITUX®; Bristol-Myers Squibb/Lilly), panitumumab (VECTIBIX®; Amgen),or zalutumumab (Genmab). The EGFR inhibitor may also be a small moleculeinhibitor. Examples of EGFR inhibitors include, but are not limited to,reversible and irreversible inhibitors, such as erlotinib (TARCEVA®;Genentech/Astellas Oncology), AZD9291 (AstraZeneca), gefitinib (IRESSA®;AstraZeneca), icotinib (BPI-2009H; Beta Pharma), rociletinib (CO-1686,AVL-301; Clovis Oncology), poziotinib (NOV120101, HM781-36B; HanmiPharmaceuticals/Spectrum Pharmaceuticals), afatinib (BIBW2292;Boehringer Ingelheim), pelitinib (EKB-569; Wyeth Pharmaceuticals),ASP8273 (Astellas), Luminespib (AUY922; Vernalis/Novartis), and XL647(Exelixis).

In some embodiments, provided herein is a method of treating lung SCC ina subject based on the presence of a HRAS mutation and resistance to anEGFR inhibitor. In some embodiments, the lung SCC can be HPV negativelung SCC. In some embodiments, the lung SCC can be HPV positive lungSCC. In some embodiments, the lung SCC can be relapsed/recurrent lungSCC. In some embodiments, the lung SCC can be metastatic lung SCC. Themethod provided herein includes (a) determining the presence or absenceof a HRAS mutation in a sample from the subject, and subsequently (b)administering a therapeutically effective amount of an FTI to thesubject if the sample is determined to have a HRAS mutation. The methodprovided herein includes (a) determining the presence or absence of aHRAS mutation in a sample from the subject, (b) determining resistanceto an EGFR inhibitor, and subsequently (c) administering atherapeutically effective amount of an FTI to the subject if the sampleis determined to have a HRAS mutation and be resistant to an EGFRinhibitor. The sample can be a tumor sample. In some embodiments, themethods include (a) determining a lung SCC patient to have a HRASmutation, and subsequently (b) administering a therapeutically effectiveamount of an FTI to the subject. In some embodiments, the methodsinclude (a) determining a lung SCC patient to have a HRAS mutation, and(b) determining resistance to an EGFR inhibitor, and, subsequently (c)administering a therapeutically effective amount of an FTI to thesubject. In some embodiments, the FTI is tipifarnib. In certainembodiments, said FTI is administered in combination with chemotherapy.In certain embodiments, said chemotherapy comprises a platinum-basedtherapy, a taxane, or a combination thereof.

5.4. Exemplary FTIs and Dosages

In some embodiments, provided herein are methods to treat SCCHN in asubject with tipifarnib or selecting SCCHN patients for tipifarnibtreatment based on the presence of a HRAS mutation. In some embodiments,the methods include treating the subject with another FTI describedherein or otherwise known in the art. In some embodiments, the FTI isselected from the group consisting of tipifarnib, arglabin, perrilylalcohol, lonafarnib (SCH-66336), L778123, L739749, FTI-277, L744832,CP-609,754, R208176, AZD3409, and BMS-214662.

In some embodiments, the FTI is administered orally, parenterally,rectally, or topically. In some embodiments, the FTI is administeredorally. In some embodiments, tipifarnib is administered orally,parenterally, rectally, or topically. In some embodiments, tipifarnib isadministered orally.

In some embodiments, the FTI is administered at a dose of 1-1000 mg/kgbody weight. In some embodiments, the FTI is administered twice a day.In some embodiments, the FTI is administered at a dose of 200-1200 mgtwice a day. In some embodiments, the FTI is administered at a dose of600 mg twice a day. In some embodiments, the FTI is administered at adose of 900 mg twice a day. In some embodiments, tipifarnib isadministered at a dose of 1-1000 mg/kg body weight. In some embodiments,tipifarnib is administered twice a day. In some embodiments, tipifarnibis administered at a dose of 200-1200 mg twice a day. In someembodiments, tipifarnib is administered at a dose of 600 mg twice a day.In some embodiments, tipifarnib is administered at a dose of 900 mgtwice a day.

In some embodiments, the FTI is administered in treatment cycles. Insome embodiments, the FTI is administered in alternative weeks. In someembodiments, the FTI is administered on days 1-7 and 15-21 of a 28-daytreatment cycle. In some embodiments, the FTI is administered orally ata dose of 900 mg twice a day on days 1-7 and 15-21 of a 28-day treatmentcycle. In some embodiments, tipifarnib is administered in treatmentcycles. In some embodiments, tipifarnib is administered in alternativeweeks. In some embodiments, tipifarnib is administered on days 1-7 and15-21 of a 28-day treatment cycle. In some embodiments, tipifarnib isadministered orally at a dose of 900 mg twice a day on days 1-7 and15-21 of a 28-day treatment cycle.

In some embodiments, the FTI is administered for at least 3 cycles. Insome embodiments, the FTI is administered for at least 6 cycles. In someembodiments, the FTI is administered for up to 12 cycles. In someembodiments, the FTI is administered orally at a dose of 900 mg twice aday on days 1-7 and 15-21 of a 28-day treatment cycle for at least threecycles. In some embodiments, tipifarnib is administered for at least 3cycles. In some embodiments, tipifarnib is administered for at least 6cycles. In some embodiments, tipifarnib is administered for up to 12cycles. In some embodiments, tipifarnib is administered orally at a doseof 900 mg twice a day on days 1-7 and 15-21 of a 28-day treatment cyclefor at least three cycles.

In some embodiments, provided herein is a method of treating a SCCHN ina subject with tipifarnib based on the presence of a HRAS mutation. Insome embodiments, the SCCHN can be HPV negative SCCHN. In someembodiments, the SCCHN can be HPV positive SCCHN. In some embodiments,the SCCHN can be relapsed/recurrent SCCHN. In some embodiments, theSCCHN can be metastatic SCCHN. The method provided herein includes (a)determining the presence or absence of a HRAS mutation in a sample fromthe subject, and subsequently (b) administering a therapeuticallyeffective amount of a tipifarnib to the subject if the sample isdetermined to have a HRAS mutation. The sample can be a tumor sample. Insome embodiments, the methods include (a) determining a SCCHN patient tohave a HRAS mutation, and subsequently (b) administering atherapeutically effective amount of a tipifarnib to the subject. In someembodiments, the methods include administering the subject with anotherFTI described herein or otherwise known in the art. In some embodiments,the FTI is selected from the group consisting of tipifarnib, arglabin,perrilyl alcohol, lonafarnib (SCH-66336), L778123, L739749, FTI-277,L744832, CP-609,754, R208176, AZD3409, and BMS-214662.

In some embodiments, the methods include (a) determining a SCCHN patientto be resistant to an EGFR inhibitor and to have a HRAS mutation, andsubsequently (b) administering tipifarnib to the subject, wherein thetipifarnib is administered orally at a dose of 900 mg twice a day ondays 1-7 and 15-21 of a 28-day treatment cycle. In some embodiments, theSCCHN patient has relapsed/refractory SCCHN. In some embodiments, theSCCHN patient has HPV negative SCCHN. In some embodiments, the SCCHNpatient has HPV positive SCCHN.

In some embodiments, the methods further comprise administering a secondtherapy to the patient having SCCHN with a HRAS mutation. In someembodiments, the second therapy is a chemotherapy, such as cisplatin,5-FU, carboplatin, paclitaxel, or platinum-based doublet (e.g.,cisplatin/5-FU or carboplatin/paclitaxel). In some embodiments, thesecond therapy is taxanes and/or methotrexate. In some embodiments, thesecond therapy is a radiation therapy. In some embodiments, the secondtherapy include those targeting PI3K pathway: BKM120 (buparlisib),BYL719, Temsirolimus, Rigosertib; those targeting MET pathway:Tivantinib, Ficlatuzumab; those targeting the HER3 pathway, Patritumab;those targeting FGFR pathway: BGJ398; those targeting CDK4/6-cell cyclepathway: Palbociclib, LEE011; RTK inhibitor: Anlotinib and chemotherapy:Oral Azacitidine. In some embodiments, the second therapy is animmunotherapy, such as anti-PD1 or anti-PDL1 antibodies. In someembodiments, the second therapy is a taxane.

In some embodiments, provided herein is a method of treating a lung SCCin a subject with tipifarnib based on the presence of a HRAS mutation.In some embodiments, the lung SCC can be HPV negative lung SCC. In someembodiments, the lung SCC can be HPV positive lung SCC. In someembodiments, the lung SCC can be relapsed/recurrent lung SCC. In someembodiments, the lung SCC can be metastatic lung SCC. The methodprovided herein includes (a) determining the presence or absence of aHRAS mutation in a sample from the subject, and subsequently (b)administering a therapeutically effective amount of a tipifarnib to thesubject if the sample is determined to have a HRAS mutation. The samplecan be a tumor sample. In some embodiments, the methods include (a)determining a lung SCC patient to have a HRAS mutation, and subsequently(b) administering a therapeutically effective amount of a tipifarnib tothe subject. In some embodiments, the methods include administering thesubject with another FTI described herein or otherwise known in the art.In some embodiments, the FTI is selected from the group consisting oftipifarnib, arglabin, perrilyl alcohol, lonafarnib (SCH-66336), L778123,L739749, FTI-277, L744832, CP-609,754, R208176, AZD3409, and BMS-214662.

In some embodiments, the methods include (a) determining a lung SCCpatient to be resistant to an EGFR inhibitor and to have a HRASmutation, and subsequently (b) administering tipifarnib to the subject,wherein the tipifarnib is administered orally at a dose of 900 mg twicea day on days 1-7 and 15-21 of a 28-day treatment cycle. In someembodiments, the lung SCC patient has relapsed/refractory lung SCC. Insome embodiments, the lung SCC patient has HPV negative lung SCC. Insome embodiments, the lung SCC patient has HPV positive lung SCC.

In some embodiments, the methods further comprise administering a secondtherapy to the patient having lung SCC with a HRAS mutation. In someembodiments, the second therapy is a chemotherapy, such as cisplatin,5-FU, carboplatin, paclitaxel, or platinum-based doublet (e.g.,cisplatin/5-FU or carboplatin/paclitaxel). In some embodiments, thesecond therapy is taxanes and/or methotrexate. In some embodiments, thesecond therapy is a radiation therapy. In some embodiments, the secondtherapy include those targeting PI3K pathway: BKM120 (buparlisib),BYL719, Temsirolimus, Rigosertib; those targeting MET pathway:Tivantinib, Ficlatuzumab; those targeting the HER3 pathway, Patritumab;those targeting FGFR pathway: BGJ398; those targeting CDK4/6-cell cyclepathway: Palbociclib, LEE011; RTK inhibitor: Anlotinib and chemotherapy:Oral Azacitidine. In some embodiments, the second therapy is animmunotherapy, such as anti-PD1 or anti-PDL1 antibodies. In someembodiments, the second therapy is a taxane.

6. Examples

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention. All of the references cited to herein areincorporated by reference in their entireties.

Example I Tipifarnib Clinical Trial in Solid Tumor Patients StratifiedBased on HRAS Mutation

A Phase 2 clinical trial was initiated to use tipifarnib in thetreatment of locally advanced unresectable or metastatic, relapsedand/or refractory, non-hematological malignancies with a known HRASmutation. Second objectives include safety and tolerability of saidmalignancies. The first exploratory objective is to explore theantitumor activity in terms of progression free survival (PFS) andduration of response (DOR) of tipifarnib in said malignancies. Thesecond exploratory objective is to explore the feasibility of collectingarchival biopsies and analyzing these biopsies for the detection oftissue biomarkers potentially related to tipifarnib activity.

The clinical trial design includes enrolling 2 cohorts of 18 patientseach. Cohort 1 enrolls subjects with malignant thyroid tumors with HRASmutations, independent of thyroid histology. Cohort 2 enrolls, in stage1, any subject with a non-hematological HRAS mutant tumor other thanthyroid cancer who meets eligibility criteria, and in stage 2, head andneck squamous cell carcinomas (SCCHN/HNSCC). Based on the anti-tumoractivity observed during stage 1 of cohort 2, the protocol was amendedto restrict enrollment in stage 2 of Cohort 2 to subjects with SCCHNwith HRAS mutations only.

This clinical trial was designed to include two stages, with the firststage including 11 evaluable patients, and the second stage including 7additional evaluable patients, and a cohort would not proceed to thesecond stage if one or no objective response is observed in a cohort inthe first stage. The clinical trial is considered positive if at least 4responses are observed in a cohort out of 18 subjects. The primaryendpoint is objective response rate, and tumor response assessments areconducted according to the Response Evaluation Criteria in Solid Tumorsversion 1.1 criteria (confirmation of response is required).

According to the protocol, tipifarnib is administered to enrolledpatients at a starting dose of 900 mg, orally with food, twice a day(b.i.d.) for 7 days in alternating weeks (Days 1-7 and 15-21) in 28 daycycles. In the absence of unmanageable toxicities, subjects may continueto receive tipifarnib treatment for up to 12 months in the absence ofdisease progression and unmanageable toxicity. Treatment may continuebeyond 12 months upon agreement of the Investigator and Sponsor. At thediscretion of the investigator, the dose of tipifarnib can be increasedto 1200 mg b.i.d. if the subject has not experienced dose limitingtoxicities at the 900 mg dose level.

Tumor assessments are performed at screening and approximately every 8weeks for the first 6 months (cycles 2, 4, 6) and then every 12 weeks(cycles 9, 12, 15, etc.) until disease progression, starting at the endof cycle 2.

Subjects who develop serious adverse events (SAE) or ≥grade 2treatment-emergent adverse events (TEAE) that are deemed related totipifarnib and lasting ≥14 days will not undergo dose escalation.Stepwise 300 mg dose reductions to control treatment-related,treatment-emergent toxicities are also allowed.

Subjects who develop serious adverse events (SAE) or ≥grade 2treatment-emergent adverse events (TEAE) that are deemed related totipifarnib and lasting ≥14 days will not undergo dose escalation.Stepwise 300 mg dose reductions to control treatment-related,treatment-emergent toxicities are also allowed.

Study Assessments:

Screening. As part of the screening procedures, all study subjectsundergo the following: Informed Consent Form (ICF) completion,evaluation of inclusion/exclusion criteria, collection of tumor HRASstatus information, medical history including the outcome and durationof response to the prior last anticancer therapy, complete physicalexamination, including weight and vital signs, use of concomitantmedications, adverse event assessment, 12-lead ECG, ECOG performancestatus, standard laboratory panels including hematology, chemistry,coagulation, and urinalysis, pregnancy test in women with childbearingpotential, and baseline tumor imaging. Serum tumor burden markers mayalso be collected at this point if deemed of interest by theInvestigator. Collection of thyroglobulin and anti-thyroglobulinantibodies for differentiated thyroid cancer and calcitonin and CEA formedullary thyroid cancer is recommended.

Treatment Phase. The evaluations to be performed on Day 1 (+/−2 days) ateach treatment cycle include: Symptom based physical examination,12-lead ECG (Cycle 1 only), ECOG performance status, standard laboratorypanels, pregnancy test in women with childbearing potential, andassessment of concomitant medications and adverse events. Tumor imagingwill be repeated approximately every 8 weeks for the first 6 months(cycles 2, 4, 6) and then every 12 weeks (cycles 9, 12, 15, etc.) untilevidence of disease progression, starting at end of cycle 2 (Day 22+/−5days) or more frequently if deemed necessary by the Investigator. Bloodsamples for the assessment of serum tumor burden markers will also becollected at the same time points as tumor imaging assessments ifsamples were previously collected during screening procedures.

End of Treatment Visit. An End of Treatment Visit takes place withinapproximately 30 days from the last dose of trial treatment orimmediately before the initiation of any other anticancer therapy,whichever occurs first. Subjects have a complete physical examination,ECOG performance status, a 12-lead ECG, standard laboratory panels,pregnancy test for women of childbearing potential, and an assessment ofconcomitant medications and adverse events. Further safety follow up isscheduled in the absence of recovery from treatment-related adverseevents.

Additional Follow Up. Tumor imaging and samples for serum tumor burdenmarker assessment continue to be repeated in intervals of approximately8 weeks for the first 6 months (cycles 2, 4, 6) and then every 12 weeks(cycles 9, 12, 15, etc.) until evidence of disease progression. Upondisease progression, subjects are contacted for survival and use ofother anticancer treatments every 12 weeks until either death or 12months after accrual in the subject's study cohort has been completed.

Example II Objective Responses with Tipifarnib in Squamous Head and NeckCarcinoma with HRAS Mutations after Failure of Cetuximab Treatment

Three subjects with a diagnosis of squamous cell carcinoma of the headand neck carcinoma (SCCHN) were enrolled in the exploratory open labelphase 2 study of Example I. Subject 1 is a 77 year old white male with adiagnosis of nasal cavity cylindrical cell carcinoma/transitional cellcarcinoma and SCCHN of the trachea as second primary who joined thetipifarnib study upon local relapse after prior paclitaxel, carboplatinand cetuximab therapy. The best response to cetuximab therapy withchemotherapy for this subject was disease stabilization. Subject 2 is a21 year old white male with a maxilla SCC diagnosis and oral cavitySCCHN as second primary and lung metastasis. Subject 3 is a 59 year oldwhite male with an oral cavity SCCHN. Subject 2 and 3 received cetuximabtherapy alone or in combination with chemotherapy, respectively, subject2 with a short-lived progression (2 cycles), followed by progressivedisease, and subject 3 with a best response of progressive disease. Nextgeneration tumor sequencing revealed the presence the Q22K HRAS mutationin the tumor of subject 1 and the Q61K HRAS mutation in subjects 2 and3. No CASP8, TP53, or PIK3CA mutations were found. The tumor of subject2 carries also a MAPK1 E322K mutation as well as point mutations inABL1, NOTCH3, RET and ROS1. HPV status for these subjects is pending.

As part of the phase 2 tipifarnib trial, subjects received treatmentwith tipifarnib at a starting dose of 900 mg, po, bid daily on days 1-7and 15-21 of 28-day treatment cycles. Subject 1 received treatment with900 mg bid for 11 cycles at which point his dose of tipifarnib wasreduced to 600 mg bid due to the onset of NCI CTCAE 4.03 grade 2peripheral neuropathy. He continues on treatment and has been currentlyover one year on study. Subject 2 was dose reduced to 600 mg bid andthen 300 mg bid during cycle 1 due to grade 2 peripheral neuropathy andcontinued on treatment for 6 additional cycles until symptomaticdeterioration/subject withdrawal at cycle 7. Subject 3 received 8 cyclesof treatment with the 900 mg bid dose and continues on study. Othertoxicities observed in these subjects were consistent with the overallsafety profile previously reported for tipifarnib (Mesa. Expert RevAnticancer Ther. 2006; 6:313-90).

Tumor assessments were performed at subject screening and approximatelyevery 8 weeks for the first 6 months (cycles 2, 4, 6) and then every 12weeks (cycles 9, 12, 15, etc.) until disease progression, starting atthe end of Cycle 2. Additional tumor assessments could be conducted ifdeemed necessary by the Investigator or for a confirmation of anobjective response. Both subject 1 and 3 experienced confirmed objectivepartial responses according to RECIST 1.1 criteria. Responses metcriteria after 6 and 2 cycles of treatment, respectively. Subject 2experienced disease stabilization with a minor 8% regression. His lasttumor scan on study did not meet imaging criteria for diseaseprogression, but he left the study after seven months of diseasestabilization due to symptomatic deterioration. CT scans of the tumorresponse in patient 1 at baseline and at cycle 4, day 22, are shown inFIG. 1A-B.

In summary, reported herein are the outcomes of three subjects withadvanced HRAS mutant SCCHN who received meaningful clinical benefit fromtipifarnib therapy. HRAS has been known to play a more prominent rolethan other RAS species in SCCHN, particularly in oral cavity tumors andthose that are HPV negative (Saranath et al. Br J Cancer. 1991;63:573-578; Anderson et al. J Otolaryngol. 1992; 21:321-326; Anderson etal. Arch Otolaryngol Head Neck Surg. 1994; 120:755-760). Overall,approximately 5% of SCCHN carcinomas but up to 16% have been reported inHPV negative oral carcinoma (Nat Commun. 2013; 4:2873). A recentcomprehensive genomic characterization of SCCHN by the Cancer GenomeAtlas Network (Nature 517, 576-582, 2015) revealed the existence of asubgroup of oral cavity tumors with infrequent copy number alterationsin conjunction with activating mutations of HRAS or PIK3CA, coupled withinactivating mutations of CASP8, NOTCH1 and TP53. According to thisgroup, the three-gene constellation of wild-type TP53 with mutant HRASand/or CASP8 may constitute an alternative pathway to tumorigenesis.

Cetuximab is currently approved for use as front line treatment of SCCHNin combination with chemotherapy or radiation or in the second linesetting as single agent treatment after failure of platinum basedtherapy. No restriction or recommendations for use in SCCHN existaccording to RAS gene status. In our study, subject 1 had a bestresponse of stable disease to his last prior anti-cancer regimen(combination of chemotherapy and cetuximab) whereas subjects 2 and 3were refractory to prior cetuximab monotherapy or in combination withchemotherapy, respectively. Of interest, subjects 2 and 3 had oralcavity tumors that carry the Q61K hotspot mutation whereas subject 1 hadthe uncommon Q22K mutation. It is unclear whether the differentialoutcome to cetuximab treatment could be in part related to histology orHRAS mutation type.

Tipifarnib is a potent and selective FTI. Phase II and III trials ofthis agent as monotherapy for solid tumors have been disappointing whilesome promising activity was observed in patients with myelodysplasticsyndrome and acute myeloid leukemia (Mesa. Expert Rev Anticancer Ther.2006; 6:313-90). Likewise, a prior study of another FTI, lonafarnib, inpatients recurrent SCCHN after platinum-based therapy was closed atinterim analysis due to the absence of objective responses (Hanrahan etal. Am J Clin Oncol. 2009; 32:274-9). Our results strongly support thehypothesis that tumor HRAS mutation may contribute to the identificationof SCCHN patients who could benefit from tipifarnib therapy. HRASmutations may also drive primary or acquired resistance to treatmentwith standard of care cetuximab based therapy. Further investigation ofthese hypotheses is warranted.

Example III Efficacy Experiments Performed with Tipifarnib inPatient-Derived Xenograft Model of HRAS-Mutant Human Head and NeckSquamous Cell Carcinoma

Experimental Methods and Procedures. Tumor fragments from stock miceinoculated with selected primary human head and neck cancer tissues wereharvested and used for inoculation into BALB/c nude mice. Each mouse wasinoculated subcutaneously at the right flank with primary human head andneck cancer model HN1420 fragment (R4P5, 2-4 mm in diameter) for tumordevelopment on day −26. HN1420 has the A146P HRAS mutation and wildtypeTP53 and is resistant to cetuximab. The mice were grouped when theaverage tumor size reached about 224 mm³ on day 0. Mice were allocatedrandomly into 2 experimental groups according to their tumor sizes. Eachgroup consisted of 3 mice, 3 mice per cage. The day was denoted as day0. The test articles were administered to the tumor-bearing mice fromday 1 through day 21 with the schedule of twice-daily (BID)×21 accordingto predetermined regimen shown in Table 1.

TABLE 1 Study design Dose Level Dose Dosing Group N Treatment (mg/kg)Route Frequency 1 3 Vehicle — p.o. BID × 21 2 3 Tipifarnib (R115777) 80p.o. BID × 21 Note: N: animal number per group. BID dosing interval was6-8 h apart.

Tumor size was measured twice weekly in two dimensions using a caliper,and the volume is expressed in mm³ using the formula: TV=0.5 a×b², wherea and b are the long and short diameters of the tumor, respectively. Thetumor size is then used for calculations of tumor growth inhibition(TGI) and T/C, as described below:

Tumor growth inhibition, % TGI=(1−(T_(i)−T₀)/(V_(i)−V₀))*100; T_(i) asthe mean tumor volume of the treatment group on the measurement day; Toas the mean tumor volume of the treatment group at day 1; V_(i) as themean tumor volume of control group at the measurement day; V₀ as thetumor volume of the control group at day 1.

The T/C value (%) is an indicator of tumor response to treatment, andone of commonly used anti-tumor activity endpoint; T and C are the meantumor volume of the treated and control groups, respectively, on a givenday.

Summary statistics, including mean and the standard error of the mean(SEM), are provided for the tumor volume of each group at each timepoint. Statistical analysis of difference in tumor volume among thegroups at study termination was conducted using an independent samplet-test. All data were analyzed using SPSS 16.0. P<0.05 was considered tobe statistically significant.

Results Summary and Discussion. The efficacy of Tipifarnib (R115777) wasevaluated in the treatment of HUPRIME® head and neck cancer xenograftmodel HN1420 in female BALB/c nude mice.

In group 1 (vehicle, p.o., BID×21) and group 2 (Tipifarnib, 80 mg/kg,p.o., BID×21), the body weight change at study termination, 1 week afterthe last dose, was 6.95% and 1.12%, respectively (not shown). There wereno animal deaths, significant body weight loss, or dosing holidaysduring the study. Thus, the test compound Tipifarnib was well toleratedin the HN1420 tumor-bearing mice.

As shown in Table 2, tumor sizes rapidly increased in thevehicle-treated animals, reaching an average of around 650 mm³ after oneweek, over 1000 mm³ within two weeks and over 1700 mm³ by the end of thestudy. By contrast, tumors in animals receiving tipifarnib remainedessentially unchanged for the first two weeks and only increased in sizeby an average of 150 mm³ during the four week course of the experiment.

TABLE 2 HN1420 Tumor Sizes in the Different Treatment Groups TumorVolume (mm³) Tipifarnib Days Vehicle (80 mg/kg, BID × 21 0 221.47 ±22.44 226.25 ± 8.07  3 398.19 ± 27.34 264.11 ± 3.31  7 647.63 ± 81.08242.79 ± 26.04 10 851.50 ± 73.06 245.81 ± 12.09 14 1025.49 ± 64.10 240.97 ± 45.13 17 1207.08 ± 53.97  227.39 ± 25.57 21 1494.10 ± 88.38 239.26 ± 44.42 24 1660.68 ± 116.57 306.90 ± 45.74 28 1720.98 ± 115.39376.10 ± 54.92 Note: data expressed as Mean ± SEM.

As shown in FIG. 2, the mean tumor size of the vehicle treated micereached 1494.1 mm3 on day 21. Tumor volume stabilization was achieved inTipifarnib treated mice with TGI of 99% and T/C of 16% (P<0.001). Theresults of tumor sizes in different groups at different time pointsafter treatments are shown in Table 3.

TABLE 3 Antitumor Activity of Tipifarnib in the Treatment of HUPRIME ®Head and Neck Cancer Xenograft Model HN1420 Tumor size (mm³)^(a) Tumorsize (mm³)^(a) TGI TIC Treatment on day 0 of treatment on day 21 oftreatment (%) (%) P value^(b) G1 Vehicle 221.47 ± 22.44 1494.10 ± 88.38— — — G2 Tipifarnib 226.25 ± 8.07   239.26 ± 44.42 99 16 <0.001 Note:^(a)Mean ± SEM; ^(b)Compared with the vehicle by independent samplet-test.

In summary, Tipifarnib (R115777) produced significant anti-tumoractivity against the primary HUPRIME® head and neck cancer xenograftmodel HN1420 in this study.

Example IV Efficacy Experiments Performed with Tipifarnib inPatient-Derived Xenograft Model of HRAS-Mutant Lung Squamous CellCarcinoma

Experimental Methods and Procedures. Tumor fragments from stock miceinoculated with selected primary human NSCLC tissues were harvested andused for inoculation into BALB/c nude mice. Each mouse was inoculatedsubcutaneously at the right flank with primary human NSCLC model LU1513fragment (R4P6, 2-4 mm in diameter) for tumor development. LU1513 hasthe Q61K HRAS mutation and the V216M TP53 mutation and is resistant tocetuximab. The mice were grouped when the average tumor size reachedabout 212 mm³ after 55 days. Mice were allocated randomly into 2experimental groups according to their tumor sizes. Each group consistedof 3 mice, 3 mice per cage. The day was denoted as day 0. The testarticles were administered to the tumor-bearing mice from day 1 throughday 21 with the schedule of twice-daily (BID)×21 according topredetermined regimen also used for the HN1420 model and shown in Table1 above. Tumor size was measured twice weekly in two dimensions using acaliper, and the volume is expressed in mm³ using the formula: TV=0.5a×b², where a and b are the long and short diameters of the tumor,respectively. The tumor size is then used for calculations of TGI, T/C,as described above in Example III. Statistical analyses were performedand interpreted as for the HN1420 model.

Results Summary and Discussion. The efficacy of Tipifarnib (R115777) wasevaluated in the treatment of HUPRIME® NSCLC xenograft model LU1513 infemale BALB/c nude mice.

In group 1 (vehicle, p.o., BID×21) and group 2 (Tipifarnib, 80 mg/kg,p.o., Bid×21), the body weight change at study termination was −5.13%and −0.54%, respectively (FIG. 3). There has been no animal death,significant body weight loss, or dosing holiday during the study. Thus,the test compound Tipifarnib was well tolerated in the LU1513tumor-bearing mice.

As shown in Table 4, tumor sizes increased quite rapidly in thevehicle-treated animals, reaching an average of around 375 mm³ after oneweek, over 500 mm³ within two weeks and over 1000 mm³ by the end of thestudy. By contrast, tumors in animals receiving tipifarnib barely grewat any time during the course of the experiment; indeed, after 28 daysaverage tumor size was slightly smaller than on day 1. Among theindividual tumor-bearing animals, one tumor had modestly increased insize, one remained static and a third regressed by approximately 75%.

TABLE 4 LU1513 Tumor Sizes in the Different Treatment Groups TumorVolume (mm³) Tipifarnib (R115777), Days Vehicle 80 mg/kg, BID × 21 0224.40 ± 15.45 198.64 ± 28.54 4 257.86 ± 29.70 209.42 ± 36.66 7 372.40 ±11.63 212.62 ± 38.76 11 420.36 ± 26.88 179.72 ± 43.67 14 509.08 ± 26.34150.11 ± 36.87 18 677.45 ± 19.63 158.82 ± 49.17 21 788.69 ± 14.38 175.78± 56.99 25  850.24 ± 104.73 154.50 ± 53.59 28 1058.26 ± 152.35 189.94 ±70.66 Note: data expressed as Mean ± SEM.

As shown in FIG. 2, the mean tumor size of the vehicle treated micereached 1058.26 mm³ at study termination. Tumor volume stabilization wasachieved in Tipifarnib treated mice with TGI of 101% and T/C of 18%(P=0.007). The results of tumor sizes in different groups at differenttime points after treatments are shown in the Table 5.

TABLE 5 Antitumor Activity of Tipifarnib in the Treatment of HUPRIME ±NSCLC Xenograft Model LU1513 Tumor size (mm³)^(a) Tumor size (mm³)^(a)TGI TIC Treatment on day 0 of treatment on day 21 of treatment (%) (%) Pvalue^(b) G1 Vehicle 224.40 ± 15.45 1058.26 ± 152.35 — — — G2 Tipifarnib198.64 ± 28.54 189.94 ± 70.66 101 18 0.007 Note: ^(a)Mean ± SEM;^(b)Compared with the vehicle by independent sample t-test.

In summary, the test compound Tipifarnib (R115777) produced significantanti-tumor activity against the primary HUPRIME® NSCLC xenograft modelLU1513 in this study.

1-50. (canceled)
 51. A method of treating EGFR inhibitor-refractory lungsquamous cell carcinoma (lung SCC) in a subject, wherein the lung SCChas an HRAS mutation, comprising administering to the subject afarnesyltransferase inhibitor (FTI).
 52. The method of claim 51, whereinsaid HRAS mutation comprises an amino acid substitution at a codonselected from a group consisting of G12, G13, Q61, Q22, K117, A146, andany combination thereof.
 53. The method of claim 51, wherein said lungSCC does not have K-Ras mutation or N-Ras mutation.
 54. The method ofclaim 53, wherein said lung SCC has wild type K-Ras and wild type N-Ras.55. The method of claim 51, wherein said lung SCC is HPV negative. 56.The method of claim 51, wherein said lung SCC is at an advanced stage ormetastatic.
 57. The method of claim 51, wherein said lung SCC isrelapsed lung SCC.
 58. The method of claim 51, wherein the EGFRinhibitor is cetuximab.
 59. The method of claim 51, wherein the FTI istipifarnib. 60-75. (canceled)
 76. A method of treating an lung SCC in asubject, wherein a sample from the subject has an HRAS mutation, andwherein the subject has never been treated with an EGFR inhibitor,comprising administering a therapeutically effective amount of an FTI tosaid subject and not administering an EGFR inhibitor.
 77. The method ofclaim 76, wherein said HRAS mutation comprises an amino acidsubstitution at a codon selected from a group consisting of G12, G13,Q61, Q22, K117, A146, and any combination thereof.
 78. The method ofclaim 76, further comprising determining the presence or absence of aK-Ras mutation or a N-Ras mutation, wherein said sample does not haveK-Ras mutation or N-Ras mutation.
 79. The method of claim 78, whereinsaid sample has wild type K-Ras and wild type N-Ras.
 80. The method ofclaim 76, wherein said sample is a tissue biopsy or a tumor biopsy. 81.(canceled)
 82. The method of claim 76, wherein determining the presenceor absence of a HRAS mutation comprises: (i) analyzing nucleic acidsobtained from said sample, wherein said Ras mutation is determined bysequencing, Polymerase Chain Reaction (PCR), DNA microarray, MassSpectrometry (MS), Single Nucleotide Polymorphism (SNP) assay,denaturing high-performance liquid chromatography (DHPLC), orRestriction Fragment Length Polymorphism (RFLP) assay; or (ii) analyzingproteins obtained from said sample. 83-86. (canceled)
 87. The method ofclaim 76, wherein said lung SCC is HPV negative.
 88. The method of claim76, wherein said lung SCC is at an advanced stage or metastatic.
 89. Themethod of claim 76, wherein said lung SCC is relapsed lung SCC.
 90. Themethod of claim 76, wherein the EGFR inhibitor is cetuximab.
 91. Themethod of claim 76, wherein the FTI is tipifarnib.